Device for amplifying target nucleic acid

ABSTRACT

A device for amplifying target nucleic acid in a sample can include a planar fluidic assembly including a transparent substrate, a porous material layer on a surface of the transparent substrate, and a cover over the porous material layer and sealingly affixed to the substrate. The cover may be spaced from the porous material layer and a flow channel defined between the porous material layer and the cover. The flow channel may have a uniform cross-section from a first end to a second end. The assembly can further include an inlet in flow communication with the first end of the flow channel to introduce sample containing target nucleic acid into the flow channel, an outlet in flow communication with the second end of the flow channel, and a plurality of nucleic acid primers retained by the porous material layer at discrete regions along and within the flow channel, each of the plurality of nucleic acid primers being complementary to a portion of the target nucleic acid in the sample to enable a primer-based amplification reaction of the target nucleic acid. The porous material layer may be configured to retain, at the discrete regions and during a primer-based amplification reaction, sample introduced to the flow channel and amplified product of the amplification reaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/269,226, filed Oct. 7, 2011, which is acontinuation application of U.S. patent application Ser. No. 11/837,600,filed Aug. 13, 2007 (now U.S. Pat. No. 8,067,159), which is acontinuation application of U.S. patent application Ser. No. 10/798,857,filed on Mar. 11, 2004 (now U.S. Pat. No. 7,972,778), which is adivisional application of U.S. patent application Ser. No. 10/131,854,filed on Apr. 25, 2002 (now U.S. Pat. No. 7,459,315), which is adivisional application of U.S. patent application Ser. No. 09/563,714,filed on May 2, 2000 (now U.S. Pat. No. 6,391,559), which is adivisional application of U.S. patent application Ser. No. 08/838,262,filed Apr. 17, 1997 (now U.S. Pat. No. 6,143,496), the entire contentsof each of which are incorporated herein by reference.

U.S. patent application Ser. No. 11/837,600 is also a continuationapplication of U.S. patent application Ser. No. 10/131,854, filed onApr. 25, 2002 (now U.S. Pat. No. 7,459,315), which is a divisionalapplication of U.S. patent application Ser. No. 09/563,714, filed on May2, 2000 (now U.S. Pat. No. 6,391,559), which is a divisional applicationof U.S. patent application Ser. No. 08/838,262, filed Apr. 17, 1997(nowU.S. Pat. No. 6,143,496), the entire contents of each of which also isincorporated herein by reference.

GOVERNMENT RIGHTS

Part of the work leading to this invention was carried out with UnitedStates Government support provided under the National Institutes ofHealth CRADA contract No. A1000079. Therefore, the United StatesGovernment has certain rights in and to the present invention.

FIELD OF THE INVENTION

The present invention relates to the in vitro amplification of a segmentof nucleic acid, methods to analyze concentrations of specific nucleicacids in sample fluids, and methods for detecting amplification of atarget nucleic acid sequence. The present invention also relates tominiaturized analytical assemblies and methods of filling miniaturizedanalytical assemblies.

BACKGROUND OF THE INVENTION

Nucleic acid amplification techniques such as polymerase chain reaction(PCR), ligase chain reaction (LCR), strand displacement amplification(SDA), and self-sustained sequence replication (3SR) have had a majorimpact on molecular biology research. In particular, PCR, although arelatively new technology, has found extensive application in variousfields including the diagnosis of genetic disorders, the detection ofnucleic acid sequences of pathogenic organisms in clinical samples, thegenetic identification of forensic samples, and the analysis ofmutations in activated oncogenes. In addition, PCR amplification isbeing used to carry out a variety of tasks in molecular cloning andanalysis of DNA. These tasks include the generation of specificsequences of DNA for cloning or use as probes, the detection of segmentsof DNA for genetic mapping, the detection and analysis of expressedsequences by amplification of particular segments of cDNA, thegeneration of libraries of cDNA from small amounts of mRNA, thegeneration of large amounts of DNA for sequencing, the analysis ofmutations, and for chromosome crawling. During the next few years, PCR,other amplification methods, and related technologies are likely to findincreasing application in many other aspects of molecular biology.

Unfortunately, problems exist in the application of PCR to clinicaldiagnostics. Development has been slow due in part to: labor intensivemethods for detecting PCR product; susceptibility of PCR to carryovercontamination—false positives due to contamination of a sample withmolecules amplified in a previous PCR; and difficulty using PCR toquantitate the number of target nucleic acid molecules in a sample. Aneed exists for a simple method of quantitative analysis of targetnucleic acid molecules in a sample.

Recently, significant progress has been made in overcoming some of theproblems of clinical diagnostic nucleic acid amplification through thedevelopment of automatable assays for amplified product that do notrequire that the reaction vessel be opened, thereby minimizing the riskof carryover contamination. Most of these assays rely on changes influorescent light emission consequent to hybridization of a fluorescentprobe or probes to amplified nucleic acid. One such assay involves thehybridization of two probes to adjacent positions on the target nucleicacid. The probes are labeled with different fluors with the propertythat energy transfer from one fluor stimulates emissions from the otherwhen they are brought together by hybridization to adjacent sites on thetarget molecule.

Another assay, which is commercially available, is the “TaqMan”fluorescence energy transfer assay and kit, available from Perkin Elmer,Applied Biosystems Division, Foster City, Calif. This type of assay isdisclosed in the publication of Holland et al., Detection of specificpolymerase chain reaction product by utilizing the 5′-3′ exonucleaseactivity of Thermus aquaticus DNA polymerase, Proc. Natl. Acad. Sci.USA, Vol. 88, pp. 7276-7280, August 1991, and in the publication ofLivak et al., Oligonucleotides with Fluorescent Dyes at Opposite EndsProvide a Quenched Probe System Useful for Detecting PCR Product andNucleic Acid Hybridization, PCR Methods and Applic., 4, pp. 357-362(1995). The “TaqMan” or 5′exonuclease assay uses a single nucleic acidprobe complementary to the amplified DNA and labeled with two fluors,one of which quenches the other. If PCR product is made, the probebecomes susceptible to degradation via an exonuclease activity of Tagpolymerase that is specific for DNA hybridized to template (“TaqMan”activity). Nucleolytic degradation allows the two fluors to separate insolution which reduces quenching and increases the intensity of emittedlight of a certain wavelength. Because these assays involve fluorescencemeasurements that can be performed without opening the amplificationvessel, the risk of carryover contamination is greatly reduced.Furthermore, the assays are not labor intensive and are easilyautomated.

The TaqMan and related assays have provided new ways of quantitatingtarget nucleic acids. Early methods for quantitation relied on settingup amplification reactions with known numbers of target nucleic acidmolecules and comparing the amount of product generated from thesecontrol reactions to that generated from an unknown sample, as reviewedin the publication by Sninsky et al. The application of quantitativepolymerase chain reaction to therapeutic monitoring, AIDS 7 (SUPPL. 2),PP. S29-S33 (1993). Later versions of this method used an “internalcontrol”, i.e., a target nucleic acid added to the amplificationreaction that should amplify at the same rate as the unknown but whichcould be distinguished from it by virtue of a small sequence difference,for, example, a small insertion or deletion or a change that led to thegain or loss of a restriction site or reactivity with a specialhybridization probe, as disclosed in the publication by Becker-Andre, etal., Absolute mRNA quantification using the polymerase chain reaction(PCR). A novel approach by a PCR aided transcript titration assay(PATTY), Nucleic Acids Res., Vol. 17, No. 22, pp. 9437-9446 (1989), andin the publication of Gilliland et al. Analysis of cytokine mRNA andDNA: Detection and quantitation by competitive polymerase chainreaction, Proc. Natl. Acad. Sci. USA, Vol. 87, pp. 2725-2729 (1990).These methods have the disadvantage that slight differences inamplification efficiency between the control and experimental nucleicacids can lead to large differences in the amounts of their productsafter the million-fold amplification characteristic of PCR and relatedtechnologies, and it is difficult to determine relative amplificationrates accurately.

Newer quantitative PCR methods use the number of cycles needed to reacha threshold amount of PCR product as a measure of the initialconcentration of target nucleic acid, with DNA dyes such as ethidiumbromide or SYBR™ Green I, or “TaqMan” or related fluorescence assaysused to follow the amount of PCR product accumulated in real timemeasurements using ethidium bromide are disclosed in the publication ofHiguchi et al., Simultaneous Amplification and Detection of Specific DNASequences, BIO/TECHNOLOGY, Vol. 10, pp. 413-417 (1992). “TaqMan” assaysused to follow the amount of PCR product accumulated in real time aredisclosed in the publication of Heid et al., Real Time Quantitative PCR,Genome Research, Vol. 6, pp. 986-994 (1996), and in the publication ofGibson et al., A Novel Method for Real Time Quantitative RT-PCR, GenomeResearch, Vol. 6, pp. 995-1001 (1996). However, these assays alsorequire assumptions about relative amplification efficiency in differentsamples during the exponential phase of PCR.

An alternative method of quantitation is to determine the smallestamount of sample that yields PCR product, relying on the fact that PCRcan detect a single template molecule. Knowing the average volume ofsample or sample dilution that contains a single target molecule, onecan calculate the concentration of such molecules in the startingsample. However, to accumulate detectable amounts of product from asingle starting template molecule usually requires that two or moresequential PCRs have to be performed, often using nested sets ofprimers, and this accentuates problems with carryover contamination.

Careful consideration of the factors affecting sensitivity to detectsingle starting molecules suggests that decreasing the volume of theamplification reaction might improve sensitivity. For example, the“TaqMan” assay requires near saturating amounts of PCR product to detectenhanced fluorescence. PCRs saturate at about 10¹¹ productmolecules/microliter (molecules/μl) due in part to rapid reannealing ofproduct strands. To reach this concentration of product after 30 cyclesin a 10 μl PCR requires at least 10³ starting template molecules(10³×2³⁰/10 μl≈10¹¹/μl). Somewhat less than this number of startingmolecules can be detected by increasing the number of cycles, and inspecial circumstances even single starting molecules may be detectableas described in the publication of Gerard et al., A Rapid andQuantitative Assay to Estimate Gene Transfer into RetrovirallyTransduced Hematopoietic Stem/Progenitor Cells Using a 96-Well FormatPCR and Fluorescent Detection System Universal for MMLV-BasedProviruses, Human Gene Therapy, Vol. 7, pp. 343-354 (1996). However,this strategy usually fails before getting to the limit of detectingsingle starting molecules due to the appearance of artifactual ampliconsderived from the primers (so called “primer-dimers”) which interferewith amplification of the desired product.

If the volume of the PCR were reduced 1000-fold to ˜10 nanoliters (nl),then a single round of 30 cycles of PCR might suffice to generate thesaturating concentration of product needed for detection by the TaqManassay, i.e. 1×2³⁰ per 10 nanoliters≈10¹¹ per microliter. Attempts havebeen made to miniaturize PCR assemblies but no one has developed acost-effective PCR assembly which can carry out PCR in a nanoliter-sizedsample. Part of the problem with miniaturization is that evaporationoccurs very rapidly with small sample volumes, and this problem is madeworse by the need to heat samples to ˜90° C. during thermocycling.

In addition to potential advantages stemming from ability to detectsingle target nucleic acid molecules, miniaturization might alsofacilitate the performance of multiple different amplification reactionson the same sample. In many situations it would be desirable to test forthe presence of multiple target nucleic acid sequences in a startingsample. For example, it may be desirable to test for the presence ofmultiple different viruses such as HIV-1, HIV-2, HTLV-1, HBV and HCV ina clinical specimen; or it may be desirable to screen for the presenceof any of several different sequence variants in microbial nucleic acidassociated with resistance to various therapeutic drugs; or it may bedesirable to screen DNA or RNA from a single individual for sequencevariants associated with different mutations in the same or differentgenes, or for sequence variants that serve as “markers” for theinheritance of different chromosomal segments from a parent.Amplification of different nucleic acid sequences and/or detection ofdifferent sequence variants usually requires separate amplificationreactions with different sets of primers and/or probes. If differentprimer/probe sets were positioned in an array format so that each smallregion of a reaction substrate performed a differentamplification/detection reaction, it is possible that multiple reactionscould be carried out in parallel, economizing on time, reagents, andvolume of clinical specimen.

A need therefore exists for a device that can form and retain a samplevolume of about 10 nanoliters or less and enable amplification to beperformed without significant evaporation. A need also exists for areliable means of detecting a single starting target nucleic acidmolecule to facilitate quantification of target nucleic acid molecules.A need also exists for performing multiple different amplification anddetection reactions in parallel on a single specimen and for economizingusage of reagents in the process.

SUMMARY OF THE INVENTION

According to the present invention, methods and apparatus for performingnucleic acid amplification on a miniaturized scale are provided thathave the sensitivity to determine the existence of a single targetnucleic acid molecule. The invention also Provides analytical assemblieshaving sample retaining means which form, isolate and retain fluidsamples having volumes of from about one microliter to about onepicoliter or less. The invention also provides a method of forming fluidsamples having sample volumes of from about one microliter to about onepicoliter or less, and retaining the samples under conditions forthermocycling. The invention also provides an analytical assembly havingmeans to determine simultaneously the presence in a sample of multipledifferent nucleic acid target molecules.

According to embodiments of the invention, PCR conditions are providedwherein a single target nucleic acid molecule is confined and amplifiedin a volume small enough to produce a detectable product throughfluorescence microscopy. According to embodiments of the invention,samples of a few nanoliters or less can be isolated, enclosed andretained under thermocycling conditions, and a plurality of such samplescan be collectively analyzed to determine the existence and initialconcentration of target nucleic acid molecules and/or sequences.According to some embodiments of the invention, sample retainingchambers having volumes of about 10 picoliters or less can be achieved.

According to embodiments of the invention, methods of forming smallfluid samples, isolating them and protecting them from evaporation areprovided wherein different affinities of a sample retaining means and acommunicating channel are used to retain sample in the means while asecond fluid displaces sample from the channel. According to someembodiments of the invention, the resultant isolated samples are thensubject to PCR thermal cycling.

According to embodiments of the invention, methods are provided fordetermining the existence and/or initial concentration of a targetnucleic acid molecule in samples of about 1 microliter or less.According to some embodiments of the invention, methods are provided fora clinical diagnosis PCR analysis which can quickly and inexpensivelydetect a single target nucleic acid molecule.

According to some embodiments of the invention, sample chambers ofabout, 1 microliter or less are provided that have a greater affinityfor a sample to be retained than for a displacing fluid. The displacingfluid displaces sample from around the chambers and isolates the sampleportion retained in the chambers.

According to embodiments of the invention, nucleic acid samples areisolated, retained and amplified in microcapillary devices havingvolumes of about 100 nanoliters or less, including microcapillary tubes,planar microcapillaries and linear microcapillaries. The devices may beprovided with absolute, selective or partial barrier means.

According to embodiments of the invention, a porous or microporousmaterial retains samples of about 100 nanoliters or less, and anassembly is provided which includes a cover for sealing sample withinthe porous or microporous material.

According to embodiments of the present invention, PCR methods andapparatus are provided wherein the sensitivity of a “TaqMan”fluorescence assay can be used to enable detection of single startingnucleic acid molecule in reaction volumes of about 100 nl or less.According to the present invention, assemblies for retaining PCRreaction volumes of about 10 nl or less are provided, wherein a singletarget molecule is sufficient to generate a fluorescence-detectableconcentration of PCR product.

According to the invention, methods are provided for carrying out PCR inminute volumes, for example, 10 nl or less, which allows detection ofPCR products generated from a single target molecule using the “TaqMan”or other fluorescence energy transfer systems.

According to embodiments of the present invention, methods of detectingand quantifying DNA segments by carrying out polymerase chain reactionin a plurality of discrete nanoliter-sized samples are provided. Thepresent invention also provides methods for determining the number oftemplate molecules in a sample by conducting replicate polymerase chainreactions on a set of terminally diluted or serially smaller samples andcounting the number of positive polymerase chain reactions yieldingspecific product. The present invention is useful in detecting singlestarting molecules and for quantifying the concentration of a nucleicacid molecule in a sample through PCR. The present invention alsoprovides methods of detecting and quantifying a plurality of target DNAsequences.

The present invention also provides methods and assemblies forseparating and/or analyzing multiple minute portions of a sample offluid medium that could be useful for other applications. Applicationsof the apparatus of the invention include the separation of biologicalsamples into multiple minute portions for the individual analysis ofeach portion, and can be used in the fields of fertility, immunology,cytology, gas analysis, and pharmaceutical screening.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in connection with various embodimentsexemplified in the accompanying drawings, wherein:

FIG. 1 is an exploded view of an analytical assembly according to anembodiment of the present invention, shown in partial cutaway;

FIG. 2 is an exploded view of an analytical assembly according to anembodiment of the present invention, shown in partial cutaway,comprising sample chambers in the form of wells formed into patternedlayers on the inner surfaces of both a top plate and a bottom plate;

FIG. 3 is a cross-sectional view through a longitudinal central portionof an analytical assembly according an embodiment of the presentinvention;

FIG. 4 is a perspective view of a bottom portion of an analyticalassembly according to an embodiment of the present invention, the bottomportion comprising sample retaining means in the form of patches offluid retaining material formed on a patterned layer coated on the innersurface of a bottom plate;

FIG. 5 is a cross-sectional view through a longitudinal central portionof an analytical assembly according an embodiment of the presentinvention;

FIG. 6A is a perspective view of a bottom portion of an analyticalassembly according to another embodiment of the present invention;

FIG. 6B is an enlarged view of portion VIA shown in FIG. 6A;

FIG. 7 is a top plan view of a microcapillary analytical assemblyaccording to an embodiment of the present invention;

FIG. 8 is a histogram showing the maximum values of thefluorescein:rhodamine intensity ratio in over 100 capillary reactions ofterminally diluted genomic DNA carried out in an assembly according tothe present invention and according to a method according to theinvention; and

FIGS. 9-14 are plots of the fluorescein:rhodamine ratio along a fewrepresentative microcapillaries containing samples subject to PCR inaccordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

According to embodiments of the present invention, methods ofmanipulating a sample of fluid medium are provided. The methods compriseloading a sample of fluid medium into sample retaining means of ananalytical assembly and displacing excess sample from areas adjacent tothe portion retained by the sample retaining means. According toembodiments of the invention, sample fluid is displaced from regionsadjacent to the retained sample, without displacing the retained sample.In some embodiments, a displacing fluid is used to isolate a retainedsample, and the displacing fluid may be curable to form a retainingchamber entrapping the fluid sample retained by, the sample retainingmeans.

The assemblies of the present invention provide samples or sampleportions enclosed in a protective environment which protects the sampleor portion from evaporation and contamination. Preferably, sample isprotected from evaporation at temperatures of about 95° C. or more, forexample, at temperatures achieved during thermal cycling underconditions for PCR. The isolated, entrapped or enclosed sample orportion is preferably protected from contamination and evaporationthroughout an amplification protocol, for example, a PCR thermal cyclingprotocol.

According to some embodiments of the invention, an analytical assemblyis provided and comprises a plurality of sample chambers each confinedin at least one dimension by opposing barriers separated by a firstdimension of about 500 microns or less, preferably by 100 microns orless, and in some embodiments by about 20 microns. Means are providedfor sealing the plurality of sample chambers to prevent evaporation andcontamination of fluid sample confined within the plurality of samplechambers. Means are provided for restraining reaction product formedfrom reactions of a chemical substance restrained within the pluralityof sample chambers. According to some embodiments, means may be providedfor minimizing diffusion and substantially preventing convection ofamplification reaction product formed from reactions of the fluid samplerestrained within the plurality of sample chambers. If provided, atleast one of the means for restraining and the means for minimizingdiffusion and substantially preventing convection may preferablycomprise a patterned layer which at least partially defines theplurality of sample chambers. Preferably, the fluid sample contains atleast one target nucleic acid molecule to be amplified and constituentsfor enabling amplification of the target nucleic acid molecule. Thefluid sample is divided into a plurality of sample portions and theplurality of sample chambers are loaded which respective portions of thefluid sample. According to some embodiments of the invention, the sampleportions are in fluid communication with each other, rather than beingcompletely isolated from each other, and separated by barrier meanswhich may be in the shape of crosses, lines, semicircles, circles havingat least one opening along the arc thereof, or other geometric shapes.According to embodiments of the invention, the barrier means may definesample retaining portions or chambers of the assembly. According to someembodiments, means for minimizing diffusion and substantially preventingconvection are provided, and may comprise the herein described barriermeans. According to some embodiments, the barrier means includesphysical structures which may extend between the aforementioned opposingbarriers which are separated by 500 microns. The barrier means may forma wall or walls between the opposing barriers. The barrier means maycomprise flow restriction means. Flow restriction means may be, forexample, semi-circular walls extending from one of the opposing barrierstoward the other, and having the concave side of the semi-circle facingthe direction of fluid flow during loading, and the semicircular arc mayhave at least hole or interruption therein through which air may escapeduring fluid sample loading. According to embodiments of the invention,the first dimension, the means for restraining, and, if provided, themeans for minimizing diffusion and substantially preventing convectionare such that the reaction product of a single target nucleic acidmolecule amplified within at least one sample chamber can attain aconcentration of reaction product molecules sufficient to be detected bya homogeneous detection assay, for example a concentration of about 10¹¹product molecules per microliter (μl). Preferably, at least one of theplurality of portions is initially free of the target nucleic acidmolecule, and at least one of the plurality of portions initiallycontains at least one target nucleic acid molecule.

According to some embodiments of the invention, an analytical assemblyis provided comprising a substrate and a cover in registry with oneanother and attached to one another and having facing surfaces spaced asubstantially uniform distance apart from one another. The facingsurface of the substrate comprises a first material having a firstaffinity to a sample of fluid to be isolated, for example, an aqueousPCR solution sample. A flow-through channel is disposed between thefirst material and the cover, and at least one sample retaining means isbounded on at least one side by the first material. The first materialmay comprise, for example, a patterned layer of moderately hydrophobicmaterial, that is, a material having a surface energy of from about 30dynes/cm to about 50 dynes/cm. The first material may be deposited onthe inner surface of the substrate.

Herein, the term “affinity” is to be understood to mean a sample-holdingcapacity or sample-holding capability. The affinity of the sampleretaining means may be caused by surface energy of the materialcontacting or restraining a sample fluid, or it may be caused byelectrostatic force, flow restriction means, temperature differences orby other means. The “affinity” of a flow-through channel may be definedby the dimensions of the channel, or by the material comprising one ormore bounding surface of the channel, or by a combination of dimensionsand bounding surface properties. The sample retaining means may havedifferent affinities to retain a sample fluid and a displacing fluid.The flow-through channel may exhibit different affinities to retain asample fluid and a displacing.

According to embodiments of the invention, the first material preferablydoes not comprise an extremely hydrophobic material; for example, thefirst material preferably does not comprise a material having a surfaceenergy of less than about 20 dynes/cm. Extremely hydrophobic materialsdo not tend to be wetted by aqueous samples or by many displacing fluidssuch as mineral oil, two-part adhesives, UV-curable and cyanoacrylateadhesives, and thus such displacing fluids would tend not to completelysurround, isolate and restrain a sample (e.g. a gaseous sample) held bythe sample retaining means.

The sample retaining means is in communication with the flow-throughchannel such that sample entering the flow-through channel can reach andbe retained by the sample retaining means. The cover and first materialshould be of such properties and/or spacial relationship to allow asample fluid and a displacing fluid to enter the flow-through channel,whether by capillary action, pressure, or other force. The sampleretaining means has a second affinity to the sample of fluid medium, andthe second affinity is greater than the first affinity. The secondaffinity may be a property induced in the first material by chemical,optical, electronic, or electromagnetic means, for example. An exemplarychemical means to permanently induce an affinity may be, for example, anO₂ plasma exposure to a portion of a silicone surface to effectivelychange the affinity of the surface to retain an aqueous sample at theportion treated. Embedding ions in a surface may also be used topermanently induce an increased or decreased affinity at a location on asurface. An affinity may be temporarily induced according to someembodiments of the invention, for example, where a surface charge on asample retaining or repelling surface is induced to increase theeffective surface tension of that surface. According to some embodimentsof the invention, a temporary affinity may be reshaped, moved orrelocated during or after a sample portion is retained, for the purposeof enlarging or joining sample portions in the assembly. According toembodiments of the invention, the difference of affinities enables theretaining means to collect a portion of sample from the flow-throughchannel and to retain the portion while a second fluid medium: isintroduced to the flow-through channel; isolates sample retained by thesample retaining means; and displaces non-retained sample from adjacentto the sample retaining means.

According to embodiments of the invention, the flow-through channel isadjacent to the sample retaining means. An entrance opening is providedfor introducing sample into the flow-through channel. According toembodiments of the invention, an analytical assembly is also providedwhich contains isolated sample entrapped by a substantially immiscibledisplacing fluid.

Methods according to embodiments of the invention involve causing thedisplacing fluid to flow through the flow-through channel and displacesample from the flow-through channel without displacing sample from thesample retaining means. Such methods are accomplished according toembodiments of the invention by providing a sample retaining meanshaving a greater affinity for the sample than for the displacing fluid.Preferably, the flow-through channel has a much lower affinity for thesample than does the sample retaining means.

According to some embodiments of the invention, the sample of fluidmedium is a gaseous sample and the sample retaining means is extremelyhydrophobic, for example, having a surface energy of from about 30dynes/cm to about 10 dynes/cm. Because of the extreme hydrophobicity ofthe retaining means, displacing fluids tend to be repelled from theretaining means, and thus avoid displacing gaseous sample from theretaining means. Displacing fluids can thus entrap, retain and isolate asample within an assembly of the invention.

According to some embodiments, an organic sample is trapped in a highsurface energy displacing fluid.

Both the sample to be isolated and the displacing fluid may beintroduced into the flow-through channel by being drawn in under theinfluence of capillary forces. Pressurized loading techniques may alsobe used, but if displacing fluid is forced into the device underpressure, the pressure should not be so high as to displace sample fromthe sample retaining means. Other means of loading sample fluid and/ordisplacing fluid may be used according to the invention and includeelectrokinetic or electrostatic loading techniques, temperaturedifferentials, centrifugal force, vacuum or suction loading, magneticattraction loading of magnetic fluids, and electrophoretic or columbicforce loading. An exemplary magnetic attraction loading techniqueinvolves drawing a fluid containing magnetic particles dispersed thereininto the device under the influence of a magnetic field and usingmineral oil containing dispersed iron particles as a displacing fluidand drawing the mineral oil into a flow-through channel with a magneticfield. According to some embodiments, the sample is loaded with magneticparticles and attracted toward and held by a sample retaining meansadjacent to or including a source of a magnetic field or a materialwhich is attracted to a magnet, for, example, an iron-containingmaterial.

Preferably, the displacing fluid is substantially immiscible with thesample of fluid medium to be isolated. According to embodiments of theinvention wherein a displacing fluid is used, the displacing fluid maycomprise a flowable, curable fluid such as a curable adhesive selectedfrom the group consisting of: ultra-violet-curable and otherlight-curable adhesives; heat, two-part, or moisture activatedadhesives; and cyanoacrylate adhesives. Exemplary displacing fluidsinclude Norland optical adhesives available from Norland Products, Inc.,New Brunswick, N.J., cyanoacrylate adhesives disclosed in U.S. Pat. Nos.5,328,944 and 4,866,198, available from Loctite Corporation, Newington,Conn., resins, monomers, mineral oil, silicone oil, fluorinated oils,and other fluids which are preferably substantially non-miscible withwater. According to some embodiments, the displacing fluid May betransparent, have a refractive index similar to glass, have low or nofluorescence, have a low viscosity, and/or be curable.

Some methods according to embodiments of the invention, includingmethods of nucleic acid molecule amplification, including PCR, maycomprise isolating a sample into a plurality of discrete retained samplepoi ions, and processing and/or analyzing the portions to determineconcentrations of components and other characteristics of the sample. Tocarry out methods of the invention wherein a plurality of sampleportions are formed and isolated, assemblies are provided having aplurality of sample retaining means. Evaluating multiple portions of asample can then be used to determine characteristics of the entiresample.

While the resolution and accuracy of many analytical techniques can beimproved according to the invention by forming and analyzing a pluralityof portions of a sample, embodiments of methods of the present inventionmore generally involve manipulating a fluid sample. Manipulating maycomprise cloning a segment of DNA, for example, where the sample offluid medium comprises a polymerase chain reaction solution, and atleast one segment of DNA to be amplified. Methods of PCR according tothe invention comprise exposing isolated sample retained by the sampleretaining means to a temperature profile which causes polymerase chainreaction amplification of a target nucleic acid molecule segment withinthe sample. According to embodiments of the invention, isolated andminute PCR samples can be retained under conditions which protect thesample during temperature profiling to dehybridize double strandedpolynucleotides, anneal primers to the dehybridized molecules, and topolymerize and thus amplify the polynucleotide.

According to embodiments of the invention, PCR methods are carried outin assemblies according to the invention, and the methods includemanipulating a PCR sample which contains an effective amount of a probeor system of probes having fluorescent properties or chemical propertiesthat change upon hybridization to a nucleic acid target. According toembodiments of the invention, a normally quenched double labeledfluorescent probe is degraded upon hybridization to target DNA duringPCR and the degradation results in increased emitted light of a certainwavelength. By measuring the amount of fluorescence and thus the amountof degraded probe, methods according to embodiments of the invention canbe used to determine whether a segment of DNA has been amplified andthereby calculate the concentration of a target DNA segment that existedin the original sample before PCR. The measured fluorescence of acertain wavelength may in some cases be used according to the inventionto quantify the amount of a DNA segment which had been retained by thesample retaining means prior to exposing a retained sample to a PCRthermal cycling profile or protocol.

other detection methods may be used to determine PCR product and/orreactant concentrations or to make other quantitative or qualitativeevaluations of many types of samples. These other detection methodsinclude agglutination, turbidity, phosphorescence detection techniques,light scattering, light absorbance, fluorescence energy transfer,fluorescence quenching or dequenching, time-delayed fluorescence,chemiluminescence and calorimetric evaluation techniques.

According to some preferred embodiments of the invention, methods ofcloning a segment of DNA are provided wherein a sample containing a DNAsegment to be amplified is divided into a plurality of sample portionsand the portions are simultaneously subjected to PCR. By providingsample portions of as small as about 10 nanoliters or less, for example10 picoliters or less, single molecules of a target DNA segment to beamplified can be detected according to the invention. For PCR methodsaccording to the invention which enable amplification of a plurality ofsample portions simultaneously, the sample from which the portions arederived may comprise a polymerase chain reaction solution, at least onesegment of DNA to be amplified, and a sufficient amount of primer tocarry out a polymerase chain reaction for multiple cycles, and themethod may comprise exposing the sample portions retained by theplurality of sample retaining means to a temperature profile whichcauses polymerase chain reaction and amplification of a target DNAsegment within the portions. According to embodiments of the invention,the presence of a single strand of a target DNA segment can be detectedin a portion retained by at least one of a plurality of sample retainingmeans.

Methods according to some embodiments of the present invention comprisemanipulating a sample of fluid medium by loading the sample into ananalytical assembly having a porous sample retaining means. Poroussample retaining means according to the invention comprise a porousstructure having an exposed porous surface and a plurality of poreshaving open upper ends at the exposed porous surface. Preferably, theexposed porous surface is moderately hydrophobic yet receptive toadhesive bonding. The pores of the sample retaining means may havesubstantially the same volume and have closed lower ends which may bedefined by a substrate onto which the porous structure may be disposed.

Preferably, the ratio of pore diameter to depth is from about 2:1 toabout 10:1, for example, 4:1 for embodiments wherein a porous materialis attached to a substrate. According to embodiments of the invention,the ratio of exposed porous surface area, that is, the area of thesurface not taken up by the openings, to the area of pore openings isfrom about 4:1 to about 1:1.5, for example, a ratio of about 1:1.

The pores have a first affinity to a sample such that when the sample isdisposed upon the exposed porous surface, the sample is drawn into theplurality of pores. The assembly used according to these methods of theinvention further comprises means to displace sample from the exposedporous surface without displacing the sample from the pores. The meansto displace may comprise a displacing fluid or a displacing device suchas a coverslip pressed against the exposed surface. The assembly mayalso include means for sealing the sample within the pores to preventevaporation and contamination of the sample during heat treatment andanalysis of the sample in the pores.

Loading sample into the pores of a porous sample retaining meanscomprises contacting the sample to the exposed porous surface andretaining the sample in the plurality of pores. Due to the affinity thepores exhibit to the sample, particularly to aqueous PCR samples, thesample is drawn into the pores by capillary force and retained therein.The methods also include displacing sample from the exposed poroussurface without displacing sample from within the pores. The methods mayalso include sealing the open ends of the pores with the sample disposedtherein.

According to some embodiments of the invention, both the top and thebottom of the pores may be open for loading and then sealed.

According to embodiments of the invention, analytical assemblies areprovided for carrying out methods of manipulating a sample of fluidmedium with a porous sample retaining means. The porous sample retainingmeans in such assemblies may comprise a microchannel array, a metal,glass, ceramic, cellulosic or polymeric screen or sieve, or a materialhaving a plurality of pores formed therein, such as a substantially flatplastic disk having a plurality of pores ablated, molded, etched ordrilled therein. The pores may be treated or coated with a material toprovide an affinity to a sample. The exposed porous surface may betreated or coated with a material to render the exposed surfacemoderately hydrophobic.

According to some embodiments, the sample retaining pores have a volumeof from about 1 microliter to about 100 nanoliters or less, preferablyabout 10 nanoliters or less, and may have pore volumes of about 1picoliter or less for some applications. According to one embodiment,the pores have volumes of about 10 picoliters.

After sample is loaded into the pores of the porous sample retainingmeans, remaining sample which is not retained by the pores but ratherwhich remains on the exposed porous surface is removed or displaced.According to embodiments of the invention, a sealing means such as amicroscope slide coverslip, tape, film or a device including othercomponents, a silicon film or, device, a device having an array ofreactants, or other means is disposed on the exposed porous surface anddisplaces sample from the exposed porous surface, without displacingsample from within the pores. After displacing sample from the exposedporous surface, the displacing means may become the sealing means ifsubsequently held, adhered or attached to the porous surface, whichwould then no longer be exposed. Preferably, the sealing means comprisesa material having a second affinity for the sample which is less thanthe first affinity, for example, the sealing means comprises arelatively hydrophobic material which contacts the exposed poroussurface, and the pores are defined by a relatively hydrophilic porousmaterial.

According to embodiments of the invention using the aforementionedporous sample retaining means, the means for displacing sample and themeans for sealing may be a single cover having a hydrophobic surfacewhich contacts the porous surface. The sealing means may be glued to thepreviously exposed porous surface, glued to a substrate on which theporous retaining means is disposed, or clamped or otherwise attached tothe porous retaining means or a substrate therefor, as for example, withclips or springs. A surface of the cover, substrate or porous sampleretaining means may provide adhesive or glue properties.

According to methods of some embodiments of the invention, a targetnucleic acid molecule or segment is amplified in at least one of thepores of a porous sample retaining means. According to such embodiments,the sample to be retained by the retaining means may comprise apolymerase chain reaction solution, and at least one target segment ofnucleic acid molecule to be amplified. The method further comprisesexposing the sample sealed within the pores to a temperature profilewhich causes polymerase chain reaction amplification of the targetsegment within at least one of the pores. In some embodiments of theinvention, the sample further comprises an effective amount of afluorescent probe which fluoresces upon degradation caused by thesuccessful amplification of the target segment in the sample. Suchmethods may further comprise measuring the fluorescence emitted fromdegraded probe after the polymerase chain reaction and determiningwhether the target segment was amplified in at least one of the pores.The fluorescence measurement may be of the amount of fluorescence, thelifetime of fluorescence, or another fluorescence property.

The methods of the present invention using a porous sample retainingmeans may further comprise using measured fluorescence to quantify theamount of target nucleic acid segment which had been retained in atleast one of the pores prior to exposing retained sample portions to athermal cycling protocol. Due to the extremely small volume of samplechambers which can be achieved with a porous sample retaining means, thepresence of a single strand of the target segment can be detected in thepores. The methods may also comprise determining the initialconcentration of the target segment which had been in the sample priorto amplification.

According to yet other embodiments of the present invention, methods ofmanipulating a sample of fluid medium are also provided wherein a sampleof fluid medium is loaded into an analytical assembly comprising amicrocapillary device, for example, a microcapillary tube having aninner diameter of about 500 μm or less, preferably about 100 μm or less.Loading may comprise filling the microcapillary tube with a sample bycapillary action. Microcapillary devices provided with a sampleretaining means, for example, a hydrophobic or hydrophilic surface or anelectromagnetic or electrostatic force, may have an inner dimension orinner diameter of about 500 μm or less. Tubular or linearmicrocapillaries not provided with sample retaining means may preferablyhave an inner dimension of about 100 μm or less. Planar microcapillariesnot provided with sample retaining means, for example, comprising thespace between two facing plates, may preferably have an inner dimensionof about 20 μm or less.

According to some embodiments, a first fluid may initially be disposedinto the microcapillary device, for example, a microcapillary tube. Thefirst fluid may preferably be substantially immiscible, and morepreferably, completely immiscible with a sample fluid. Loading thencomprises disposing a portion of a sample fluid into the microcapillarytube adjacent the first fluid, and subsequently disposing a second fluidinto the microcapillary tube adjacent the sample. The second fluid isalso preferably substantially immiscible, and more preferably completelyimmiscible, with the sample. The sample is disposed between andrestrained by the first and second fluids. The first and second fluidsmay be the same fluid, and can be, for example, mineral oil or a gas ora curable monomer formulation, if the sample is an aqueous fluid. Themicrocapillary device may contain more than one isolated sample portion,which may be separated by one of the first and second fluids. Eachportion may be about 100 nl or less, preferably 10 nl or less.

Some methods of the invention which employ microcapillary sampleretaining means may also comprise sealing both ends of themicrocapillary tube with the sample therein. The sample may fill theentire capillary tube and be sealed therein, or the sample may be sealedin the tube sandwiched between first and second fluids. Means may beemployed to load numerous sample portions, such as an ink jet or fluidiccontrol apparatus.

According to embodiments of the invention, microcapillary analyticalassemblies are also provided and can be used to isolate and retain asample of fluid medium comprising about 100 nanoliters or less of afluid medium, preferably about 60 nanoliters or less. Assemblies arealso provided which comprise a plurality of such microcapillary tubes,each tube having a sample of fluid medium disposed therein. A pluralityof tubes may be attached to a pair of microscope slide coverslips, or toa tape or pair of tapes, or otherwise held together.

The microcapillary sample retaining devices of the present invention maybe used to carry out nucleic acid amplification methods, according toembodiments of the invention. When used in PCR applications, the fluidsample may comprise a polymerase chain reaction solution which mayinclude reagents, enzymes, buffer, bovine serum albumin and other wellknown ingredients commonly used to perform a polymerase chain reaction.Methods according to the invention which employ the inventivemicrocapillary devices may further comprise exposing the sample sealedwithin the microcapillary tube, or within a plurality of tubes, to atemperature profile which causes amplification of the target segmentwithin the sealed sample.

As mentioned in connection with other methods and apparatus according tothe invention, the PCR sample in the microcapillary tube may furthercomprise an effective amount of a fluorescent probe which fluorescesupon degradation of the probe caused by the successful amplification ofthe target segment. The fluorescence emitted from the hybridized probecan be measured to determine whether the target segment was amplified.By using different dilutions of starting sample or by using replicatesamples in different volumes, the concentration of starting targetsegment can be determined.

According to embodiments of the present invention wherein PCR is carriedout in a microcapillary assembly, fluorescence or other detectionproperties are promptly analyzed after PCR thermal cycling, for example,within about 5 hours, more preferably within about 1 hour after PCRthermal cycling is complete. Prompt analysis of the fluorescence emittedmaximizes the concentration of measured degraded probe along regions ofthe microcapillary tube, before the degraded probe diffuses along thetube and becomes less detectable. Preferably the amplification iscarried out as rapidly as possible, for example, in less than one hour.

Miniaturized assemblies according to embodiments of the invention havebeen briefly described above and will be discussed in greater detailbelow. Assemblies according to embodiments of the invention can takemany forms but can generally be classified into three types, (1)assemblies having flow-through channels and sample retaining means incommunication with the flow-through channel, (2) assemblies havingporous sample retaining means and means for sealing sample within sampleretaining pores, and (3) assemblies comprising at least onemicrocapillary tube.

According to embodiments of the invention, assemblies are provided formanipulating samples of fluid medium, for example, methods for isolatingsmall sample volumes, such as sample volumes of 100 nanoliters or less.

Some assemblies according to the invention comprise a substrate and acover in registry with and attached to one another and having facingsurfaces spaced a substantially uniform distance apart from one another.The facing surface of the substrate comprises a first material having afirst affinity to a sample of fluid medium to be contained in theassembly. Far example, if an aqueous fluid is to be manipulated, thefirst material, is preferably a moderately hydrophobic material. Thefirst material preferably defines at least a portion of a flow-throughchannel which is disposed between the substrate and the cover. Theflow-through channel is in fluid communication with a sample retainingmeans, and the retaining means is bounded on at least one side thereofby the first material. The sample retaining means has a second affinityto the same sample of fluid medium. Preferably, the second affinity tothe sample fluid is greater than the first affinity thereto, preferablymuch greater, enabling the sample retaining means to retain or collect aportion of a sample of fluid medium which flows through the flow-throughchannel and to retain the portion while a second fluid medium flowsthrough and displaces sample from the flow-through channel. The resultcan be a very small isolated portion of the sample being retained by thesample retaining means and entrapped, encased or otherwise surrounded bythe second fluid medium in the flow-through channel.

According to embodiments of the invention wherein the assembly comprisesa flow-through channel and sample retaining means, the substrate may bea first plate having a patterned layer of the first material formedthereon. The cover may be a second plate attached to and substantiallyparallel to the first plate, with the first and second plates havingfacing substantially parallel planar surfaces. The first and secondplates may each be rigid or flexible, flat or contoured, a sheet, afilm, a microscope slide, a microscope slide coverslip, a glass plate, atape, a device including other components, a silicon device, a siliconfilm, or the like. According to some embodiments, the plate has asubstantially planar surface on a side adjacent or defining the sampleretaining means. The patterned layer is preferably located between theplanar surfaces and at least partially forms a boundary for the sampleretaining means. For example, when the sample retaining means is achamber formed from an opening in, a cavity or recess in, or a holethrough the patterned layer, the closed end of the chamber may bedefined by the substrate surface on which the patterned layer isdisposed or by the patterned layer, and the patterned layer may definethe sidewalls of the sample chamber. According to embodiments whereinthe sample retaining means is a recessed sample chamber at leastpartially defined by the patterned layer, the chamber may comprise asidewall, a closed lower end, and an upper end which may be open orclosed. The sample chamber has a communication with the flow-throughchannel. According to some embodiments of the invention, the samplechamber also extends into and is partially defined by a patterned layerformed on the facing surface of the assembly cover, in which case thesample chamber may have a closed upper end and the communication to thechamber may be formed in the chamber sidewall, for example, an annulargap in an otherwise continuous sidewall. According to other embodimentsof the invention, the cover is substantially planar and the samplechamber does not extend into the cover or a layer disposed on the cover,in which case the chamber has an open upper end in communication withthe flow-through channel.

According to embodiments of the invention wherein the substrate andcover comprise first and second plates, the closed lower end of thesample chamber may comprise the first plate. The sample retaining meansmay comprise a third material at the lower end of the sample chamber.The third material may in some cases be a hydrophilic material depositedon the first plate and defining the lower end of the chamber. Accordingto some embodiments, the third material may be very hydrophobic. Foraqueous samples in particular, hydrophilic materials may be used at thelower end of the sample chamber. According to some embodiments of thepresent invention, a sample chamber comprising a recess in a substrateor substrate coating may preferably have hydrophilic material at a lowerend thereof and hydrophobic material forming the sidewall. Preferably,the hydrophilic lower end provides an affinity to an aqueous PCR samplewhich is sufficient to retain the sample while a displacing fluidcarries away sample adjacent the sample chamber. The hydrophobicsidewalls prevent sample from being displaced from the sample chamber.

Preferred sample chambers according some embodiments of the invention,for nucleic acid amplification methods to detect single target nucleicacid molecules, have volumes of from about 1 microliter to about 1picoliter or less. Printing, photolithography, etching, ablation andother methods of forming sample chambers in, for example, printedlayers, can provide sample chambers of ten nanoliters or less, forexample, about 100 picoliters.

Screen printing and photolithography are preferred methods of forming apatterned layer on the substrate. Screen printing methods can provide apatterned layer on a 1 inch by 3 inch microscope slide wherein the layercontains over one thousand isolated and spaced sample chambers eachhaving a volume of about 1 nanoliter. Such a patterned layer couldenable over one thousand PCR chambers of about 1 nanoliter each, all onthe surface of a 1″×3″ microscope slide. Photolithographic methods canprovide from about 10,000 to over 100,000 sample chambers of about 100picoliters each on a 1″×3″ substrate.

According to embodiments of the invention, the first material orpatterned layer material is moderately hydrophobic. Herein, the term“moderately hydrophobic” refers to a material or layer that exhibits acontact angle to water of from greater than about 30° to less than about85°. According to some embodiments, a hydrophobic patterned layer isdisposed on the facing surfaces of both the substrate and the cover, theaverage contact angle of the two patterned layers is preferably morethan about 30° to less than about 85°, and more preferably each of thetwo layers is moderately hydrophobic. Patterned layers exhibiting highercontact angles to water may be employed on the substrate surface if thecover comprises a hydrophilic facing surface. Preferably, the patternedlayer or first material is of such a nature that displacing fluid canbond thereto when the second fluid or displacing fluid is apolymerizable fluid. If the patterned layer or first material comprisesTEFLON, for example, a curable or polymerizable displacing fluid may notbe capable of bonding thereto and thus may not sufficiently seal anentrapped sample fluid posing contamination and evaporation risks.

According to some embodiments of the invention, the substrate and covercomprise first and second facing planar surfaces with a first layer, onthe first surface and a second layer on the second surface. According tosome embodiments, sample chambers are formed in the first layer and thesecond layer is substantially smooth. According to some otherembodiments, the second layer also has sample chambers formed therein,preferably mirroring the sample chambers formed in the first layer. Thesecond layer may comprise a moderately hydrophobic material.

According to some embodiments, the interior surface(s) of the substrateand/or cover may be altered by chemical or, other means to form apattern of retaining means having altered surface energy or structure.

According to some embodiments of the invention, a sample chamber,comprises a hole or recess formed in a first patterned layer, and a holeor recess formed in a second patterned layer, the sidewall comprisesboth the first and second patterned layers, a flow-through channel isdisposed between the first and second patterned layers, and thecommunication to the flow through channel interrupts the sidewall.

According to embodiments comprising first and second patterned layers,the first and second layers may comprise the same material. The secondpatterned layer may have a substantially smooth surface facing the firstpatterned layer, and the second layer may have a substantially uniformlayer thickness. In embodiments wherein a smooth and continuous secondlayer is provided on the inner surface of a cover or top plate of anassembly, the sample chamber may have an open upper end in fluidcommunication with the flow-through channel and the sidewall maycompletely comprise the first patterned layer.

The patterned layer may be the retaining means, for example, hydrophobicor hydrophilic spots on the substrate or cover. According to someembodiments, the patterned layer, may comprise the retaining means andmay comprise a microporous material such as an epoxy material which ishighly filled with micron-sized beads.

According to embodiments of the invention wherein an assembly comprisesa flow-through channel, the flow-through channel may have an entranceopening and an exit vent. Assemblies designed for forced pressureloading may not require an exit vent. In embodiments of the inventioncomprising a flow-through channel, the channel may have a substantiallyuniform cross-sectioned area throughout. In other embodiments, thecross-sectional area of the flow through channel increases or decreasesin a direction from the entrance opening to said exit vent. The changingcross-sectional area of the flow-through channel can influence thetravel of the sample and/or displacing fluid through the flow-throughchannel due to increasing or decreasing resistance of the fluid flow.For example, in embodiments wherein the flow-through channel increasesin cross-sectional area from adjacent to the entrance opening in thedirection of the exit vent, fluid flow toward the exit vent is subjectto decreased flow resistance compared to embodiments wherein thecross-sectional area is the same throughout the flow-through channel.One method of forming a flow-through channel with an increasing ordecreasing cross-sectional area is to space the cover or top platefurther from the substrate or bottom plate at the entrance end of theassembly than at the exit end. Although the substrate and cover, orfirst and second plates, would remain substantially parallel to eachother as defined by the present invention, they would not be exactlyparallel. Another method of forming an increasing or decreasingflow-through channel cross-sectional area is to form the patterned layerthicker at one end of the device than at the opposite end of the device,and to have the thickness of the layer gradually increase or decrease inthickness. According to some embodiments wherein holes in a patternedlayer define sample retaining means, the retaining means may havedifferent sizes and shapes.

According to some embodiments of the invention, a hydrophilic pattern isprovided to form sample retaining means and the pattern may be inducedby electrets or by internal or external electrodes to provide a chargedsurface having higher surface energy and wettability than a flow-throughchannel in communication with the retaining means.

According to some preferred embodiments of the invention, an analyticalassembly is provided with a plurality of sample retaining meansseparated from one another. In some embodiments, the plurality of sampleretaining means comprise sample chambers formed in a patterned layerdisposed between the substrate and cover or first and second plates. Thesample retaining means may comprise chambers each having a sidewall, anupper end, a closed lower end, and a communication with a flow-throughchannel.

Filled assemblies are also provided according to embodiments of theinvention, and may comprise a sample fluid retained by the sampleretaining means, and a different, second fluid retained in theflow-through channel. Preferably, the second fluid is substantiallyimmiscible with the sample fluid. The sample fluid may comprise apolymerase chain reaction solution and at least one segment of DNA to beamplified. The second fluid is referred to as a displacing fluid and maycomprise a curable fluid, particularly curable adhesives, and preferablyfluid adhesives selected from the group consisting of light-curable,heat-curable, two-part-curable, moisture-curable and cyanoacrylateadhesives. When UV-curable adhesives are used and cured, UV-blockingspots may be provided on the cover and/or substrate, aligned with thesample retaining means, to protect the retained sample from harmfullight.

According to some embodiments of the invention, wherein an assembly isprovided having opposing surfaces, sample retaining means between thesurfaces, and a flow-through channel in communication with the retainingmeans, the sample retaining means may comprise a fibrous or porousmaterial which absorbs a sample of fluid medium through capillaryforces. The fibrous or porous material may be formed on a patternedlayer of a first material deposited on one of the surfaces. The firstmaterial may comprise a moderately hydrophobic material. A secondmaterial layer may be included on one of the facing surfaces and maycomprise a moderately hydrophobic material. The fibrous or porousmaterial may be a cellulosic material, a filter paper material,absorbent textured material, absorbent sintered materials, absorbentpastes, microporous membranes, fiberglass, and the like. Preferably, thefibrous or porous material is a porous membrane having a maximum porediameter size of about 1 micron. The fibrous or porous material may fillthe gap between the substrate and cover or may be disposed on just oneof the interior substrate or cover surfaces.

The fibrous or porous material has a wicking rate for a sample which canbe measured in millimeters per second, and sample flowing through theflow-through channel advances through the channel at an advancing ratewhich can be measured in millimeters per second. Preferably, the wickingrate exceeds the advancing rate, thus minimizing the possibility of anaqueous sample entrapping or encircling the sample retaining meansbefore air in the retaining means can escape and be carried away by theadvancing fluid. In cases where the advancing rate exceeds the wickingrate, sample tends to be absorbed too slowly by the retaining means andthe advancing fluid in the channel tends to surround the retaining meansand entrap air before the air has a chance to escape.

The size and shape of the porous retaining means also influences whetherair will be trapped in the sample retaining means. For larger sampleretaining means, there is a greater chance that air may be entrapped inthe sample retaining means than for smaller sample retaining means.Therefore, it is preferable to use higher wicking rates for largerfibrous or porous sample retaining means than for smaller retainingmeans. For example, if the retaining means has a wicking rate of 1mm/sec and the advancing rate is also 1 mm/sec, a substantially flatsample retaining means having a diameter of about 1 mm tends to bewicked by sample without entrapping air, whereas a retaining meanshaving a diameter of about 2 mm may not be completely wicked with samplebut instead is more likely to entrap air. Retaining means that areelongated in the direction of sample flow may be preferred for largesamples.

According to some embodiments of the invention, sample retaining meansare provided which exhibit an affinity to retain a sample throughapplication of a generated force. Rather than using materials havingdifferent affinities, the sample retaining means may be provided withmeans to generate, for example, an electrostatic force. Indium oxide orother conductive coating materials can be strategically placed on or inthe substrate, cover or patterned material to form a region or spotwhich can be charged to form an electrostatic attractive force. If acurable displacing fluid is then used to displace sample from around thecharged region or spot, generation of the force is no longer neededafter the displacing fluid displaces sample and/or cures.

In some embodiments wherein a generated force is used to retain samplein a region or at a spot, the generated force may be a temperaturegradient or temperature altering means which provides a temperature tothe sample retaining means which affects the affinity of the retainingmeans to a sample, and produces a different affinity for the sample atthe retaining means than at surrounding regions of the assembly such asin the flow-through channel.

According to some embodiments of the invention having a flow-throughchannel, the sample retaining means is not a sample well or recess butrather a sample chamber formed between a patch of a second material anda cover or patch of a third material. The second and third materials maybe the same, and preferably both the second and third materials have agreater affinity for a sample of fluid medium than a first materiallayer which at least partially defines the flow-through channel. Forexample, the second and third materials preferably have a greateraffinity for an aqueous PCR sample than the affinity the first materialexhibits to the same PCR sample. According to some embodiments, thepatch or spot of the second and/or third material is disposed on thefirst material.

According to embodiments of the invention having a flow-through channel,the substrate is a first plate having a patterned layer formed thereon,and the cover is a second plate attached to and substantially parallelto the first plate. The first and second plates have facingsubstantially parallel planar surfaces, and the patterned layer islocated between the planar surfaces and at least partially bounds thesample retaining means. The sample retaining means comprises a sampleretaining patch disposed on the patterned layer and is spaced a firstdistance from the facing surface of the second plate. Preferably, theflow-through channel has a bottom surface which is spaced from thefacing surface of the second plate by a second distance, and the seconddistance is greater than the first distance. According to some preferredembodiments, the sample retaining means comprises a plurality of sampleretaining patches disposed on the patterned layer, spaced from oneanother, and spaced a first distance from the facing surface of thesecond plate.

According to some embodiments of the invention, the substrate comprisesa flexible material, for example, a polymeric film such as polypropylenefilm, polyethylene film, polycarbonate film, polyethyleneterephthalatefilm, silicone film, teflon film, celluloid film, or other film such asa metal or ceramic film. When a flexible material is used, it mayinstead be molded or formed if not in the form of a tape. The cover mayalso comprise a flexible material such as a polymeric film, such thatthe entire assembly is substantially flexible. According to suchembodiments, a flexible tape can be constructed and cut to sizedepending upon the number of sample retaining means desired to beutilized. An entrance opening and an exit vent can be used to load anddisplace sample fluid, and to load displacing fluid. The entranceopening and/or exit vent may be in the form of a gap formed between thesubstrate and cover at an end or gaps formed at opposite ends of a pieceof tape and in communication with a flow-through channel and a supply ofsample to be apportioned.

According to embodiments of the invention, a combination is providedwhich includes a miniaturized assembly for containing a sample of fluidmedium, a means for displacing a portion of a sample of fluid medium,and a sealing means, which may be packaged together as a kit oravailable separately. The miniaturized assembly comprises a substratehaving a surface and a sample retaining means disposed on the surface.The sample retaining means comprises a porous structure having a poroussurface and a plurality of pores having open ends at the surface. Thepores may each have substantially the same volume and closed lower ends.The pores have a first affinity to a sample of fluid medium such that asample of fluid medium disposed upon the porous surface is drawn intoand retained by the plurality of pores. The pores preferably have anaffinity to retain a sample of aqueous medium, particularly an aqueousPCR sample. The means to displace a sample of fluid medium from theporous surface without displacing the sample from within said pores maycomprise a cover or coverslip, for example, a standard microscope slidecoverslip. Preferably, the cover or coverslip has a hydrophobic surface,which may be adhesive, and which contacts the porous surface and is notwet by aqueous samples. The cover or coverslip may be permanentlyattached to the substrate after displacing sample from the poroussurface, or the cover or coverslip may be removed and replaced with asealing device. The sealing device according to this and otherembodiments is preferably transparent so that fluorescence emitted fromsample retained by the sample retaining means can be observed and/ormeasured. The means for displacing fluid and the means for sealing arethe same device, for example, a single covering device such as a singlecoated microscope coverslip. The porous sample retaining means may be ametal plastic, glass or ceramic sieve or screen, or other materialshaving a plurality of pores formed therein, such as a substantially flatplastic disk having a plurality of pores etched, ablated, molded,drilled, poked or otherwise formed therein. The volume of the pores maybe from about 1 microliter to about 1 picoliter or less. Preferably, thevolume of the pores is about 100 nanoliters or less, and for someapplications may be 1 nanoliter or less.

According to some embodiments of the invention, an analytical assemblyis provided comprising a microcapillary tube having opposite ends, aninner diameter of about 100 microns or less, and at least oneamplifiable nucleic acid molecule segment entrapped inside the tube.Microcapillary tubes having inner diameters of about 500 μm or less mayalso be used if a sample restraining means is included in the tube, for,example, glass beads, gels, absorbent particles, barrier means orelectrophoretic means. Tube lengths of from about 1 mm to about 100 mmare preferred.

Capillary action is preferably used to introduce a sample fluid in themicrocapillary tube, and after the sample is disposed in the tube, thetube is sealed at both ends thereof to entrap the sample inside. Thesample may be divided into a plurality of portions separated by, forexample, mineral oil. The entrapped sample or sample portions is/arethereby protected from contamination and from evaporation. The sample offluid medium disposed in the tube may have a volume of about 100nanoliters or less, more preferably about 10 nanoliters or less, and forsome applications, a volume of about 1 nanoliter or less.

According to some microcapillary embodiments, a first fluid is disposedin the tube on a side of the sample and adjacent to the sample, and thefirst fluid is preferably substantially immiscible with the sample. Asecond fluid may be disposed in the tube on the opposite side of thesample and adjacent to the sample, and the second fluid is alsopreferably substantially immiscible with the sample, such that thesample is disposed between, and restrained by, the first and secondfluids. According to some embodiments, the sample is an aqueous mediumand the first and second fluids are both mineral oil or polymerizablefluid. According to some embodiments, the sample preferably comprisesabout 100 nanoliters or less of a fluid medium. Multiple isolated samplesegments may be introduced by ink jet or other sample dispensing means.

The microcapillary assemblies according to embodiments of the inventionmay comprise a plurality of sealed microcapillary tubes having innerdiameters of 100 microns or less, with each tube containing at least oneportion of a sample of fluid medium. Microcapillary tubes having innerdiameters of about 500 μm or less may also be used if a restrainingmeans such as a mineral oil is used to separate minute sample portionsfrom each other. Tribe lengths of from about 1 mm to about 100 mm arepreferred. The tubes may each contain substantially immiscible fluids onopposite sides of the respective portion(s) of sample within each tube,such that the sample portion(s) in each tube is/are disposed between,and restrained by, the substantially immiscible fluid. The substantiallyimmiscible fluids are preferably essentially immiscible with the sample.According to some embodiments, the sample comprises a polymerase chainreaction solution including primer, and at least one segment of anucleic acid molecule to be amplified.

According to embodiments wherein microcapillary assemblies are provided,the tube or tubes used in the assembly may be self sealing or sealed ateither or both ends thereof with a curable fluid, preferably a curableadhesive. Exemplary curable adhesives include those selected from thegroup consisting of light-curable, heat-curable, two-part-curable,moisture-curable and cyanoacrylate adhesives. When UV-curable adhesivesare used and cured, UV-blocking spots may be provided on the coverand/or substrate, aligned with the sample retaining means, to protectthe retained sample from harmful light.

According to yet other embodiments of the invention, a method isprovided for loading a fluid sample into an assembly for isolating andretaining a small portion of the sample. Assemblies for carrying outsuch methods comprise a sample retaining means, for example, samplechambers, for retaining the small portion of the sample. The method ofloading a fluid into an assembly may comprise providing a first fluid tobe retained and providing a displacing fluid. An assembly is providedcomprising a substrate and a cover having facing surfaces spaced fromone another, wherein the facing surface of at least one of the substrateand the cover comprises a first material. The assembly has aflow-through channel between the substrate and the cover, and a firstfluid retaining means bounded on at least one side thereof by the firstmaterial. The channel is in communication with the flow-through channel.The first fluid retaining means has a first affinity to the first fluidand a second affinity to the displacing fluid, and the flow-throughchannel has a third affinity to the first fluid and a fourth affinity tothe displacing fluid. The first, second, third and fourth affinities aresuch that the first fluid retaining means retains at least a portion ofthe first fluid loaded into the assembly while displacing fluiddisplaces first fluid from the flow-through channel. The displacingfluid preferably can displace the first fluid from the flow-throughchannel without substantially displacing first fluid from the firstfluid retaining means. The method further comprises causing the firstfluid to be loaded into the flow-through channel and be retained by thefirst fluid retaining means, and causing the displacing fluid to enterthe flow-through channel and displace first fluid from the flow-throughchannel without substantially displacing first fluid from the firstfluid retaining means. According to some embodiments of the invention,the first affinity is greater than the second affinity. According tosome embodiments, the fourth affinity is greater than the thirdaffinity. According to some embodiments, the flow-through channelcomprises at least one bounding surface and has dimensions, and thethird and fourth affinities are provided by the dimensions of theflow-through channel and the respective affinities of the at least onebounding surface to the first fluid and to the displacing fluid.

According to some embodiments of the invention, a method is providedwhich comprises providing an assembly including a substrate and a coverin registry with and attached or affixed to one another and havingfacing surfaces spaced a substantially uniform distance apart from oneanother. The facing surface of the substrate comprises a first materialhaving a first affinity to a sample of fluid medium to be contained inthe assembly. A flow-through channel is disposed between the substrateand the cover, and the sample retaining means is bounded on at least oneside thereof by the first material. The first material may be, forexample, a surface of a glass plate or slide, or a patterned layer suchas a hydrophobic material layer deposited on the facing surface of thesubstrate. The sample retaining means is in communication with theflow-through channel and has a second affinity to the sample of fluidmedium, wherein the second affinity is greater than the first affinity.The different affinities enable the sample retaining means to collect aportion of a sample of fluid medium which flows through the flow-throughchannel and to retain the portion while a second fluid medium flowsthrough and displaces the sample from the flow-through channel.

The method of loading also comprises causing the sample to flow throughthe flow-through channel and to be retained by the sample retainingmeans, and causing a displacing fluid to flow through the flow-throughchannel and displace the sample of fluid medium from the flow-throughchannel without displacing sample from the sample retaining means,wherein the sample retaining means has a greater affinity for the samplethan for the displacing fluid. The displacing fluid thereby covers,entraps, encircles, and/or surrounds, and isolates the sample retainedby the sample retaining means on at least one side of the retainedsample.

According to some methods of loading, the displacing fluid is preferablysubstantially immiscible with the sample of fluid medium, and morepreferably, is essentially or completely immiscible with the sample.According to embodiments of the invention, the sample comprises anaqueous medium. According to embodiments of the invention, thedisplacing fluid may comprise a curable fluid, preferably a curableadhesive.

According to some methods of loading, the flow-through channel has anentrance opening for introducing sample to the channel, and an exit ventfor the escape of displaced air, sample or excess displacing fluid fromthe channel, and the method further comprises introducing the displacingfluid to the channel through the entrance opening and causing sample inthe channel to be displaced by the displacing fluid. The displacingfluid may cause excess sample to exit the assembly through the exitvent, particularly in embodiments wherein the displacing fluid is forcedinto the assembly by other than capillary forces, for example, bypressure loading. In some embodiments, the exit vent may comprise aporous or absorbent material such as paper, or other materials that arewettable by the sample.

According to some embodiments, the method further comprises sealing theentrance opening and exit vent after the displacing fluid displacessample from the channel. In some embodiments, the displacing fluid curesto seal the entrance opening and the exit vent. Preferably, thedisplacing fluid cures adjacent to the sample retaining means to sealthe sample within the sample retaining means. In some embodiments, thesample retaining means retains about 1 microliter to about 1 picoliterof sample or less, preferably about 10 nanoliters of sample or less, andfor some embodiments, about 1 nanoliter or less.

According to embodiments of the invention, methods are also provided fordetermining the existence and/or quantitation of multiple types ofnucleic acid target molecules. According to some embodiments, differentamplification targeting reagents can be loaded separately or togetherwith respective different samples to be amplified. According to someembodiments, different amplification targeting reagents are preloadedinto different sample retaining means within a single assembly, forexample, an assembly having two parallel but separated flow-throughchannels having respective sample retaining means in communication withone of the channels. According to some embodiments, each sampleretaining means contains a specific primer, pair of primers, and/orprobe and at least one of the sample retaining means contains a primeror probe that differs from the primer or probe of a second retainingmeans. A sample possibly containing more than one different targetsequence to be amplified is then introduced and retained by the sampleretaining means. When isolated, amplified and quantitated, the existenceand quantitation of two or more different target sequences can bedetermined, for example by using a fluorescence energy transfer assay.According to the invention, the device is permanently sealed with acurable adhesive and a homogeneous assay is performed. The sealed assaydoes not require physical separation of components of the assembly todetermine whether specific target sequences have been amplified.

The invention will now be described with reference to the drawingfigures which are exemplary in nature and not intended to limit thescope of the invention in any respect.

FIG. 1 shows an exploded view of an analytical assembly according to anembodiment of the present invention, shown in partial cutaway. Theassembly comprises a substrate 20 and a cover 22. In the embodimentdepicted, the substrate and cover comprise substantially rigid platessuch as microscope slides, but may comprise more flexible materials suchas microscope slide coverslips. The substrate includes a bottom plate 23having an inner surface 24, and a patterned layer 26 of a first materialdisposed on the inner surface. The first material may be a patternedlayer comprising a hydrophobic material, and provides the substrate 20with a facing surface 28 which faces the cover 22. Within the patternedlayer 26 of the first material are formed a plurality of wells or holestherethrough defining sample chambers 30. Each sample chamber 30 has aclosed lower end 32, defined by the inner surface 24 of the bottom plate23. Each sample chamber also has a sidewall which extends from theclosed lower end 32 up to the facing surface 28 of the substrate. Insome embodiments, the sample chamber extends up through the flow-throughchannel.

The cover 22 comprises a top plate 34 and a facing surface 36 which maycomprise the same material as the top plate or a patterned layer 38 of asecond material disposed on the inner surface of the top plate. Thesecond material may be the same as the first material, and in someembodiments is preferably moderately hydrophobic, that is, it preferablyhas a surface energy of from about 30 dynes/cm to about 50 dynes/cm.Exemplary materials for the second material include silanes,methacrylates, epoxies, acrylates, cellulosics, urethanes, silicones,and materials having good adhesion to the displacing fluid, for example,materials having good adhesion to displacing fluids comprising curableadhesives. The patterned layer 38 may be smooth and of uniform thicknessor it may have a plurality of sample chambers formed thereincomplementary to the chambers formed in patterned layer 26.

When assembled, the substrate 20 and cover 22 are attached together,with the facing surface 36 of the cover being spaced from the facingsurface 28 of the substrate. The space between the facing surfaces 36and 28 defines a flow-through channel through which sample fluid anddisplacing fluid may travel. The facing surfaces 36 and 28 aremaintained spaced apart by spacer strips 40, which may comprise transferadhesive strips, films or adhesive layers applied to edge regions of thesubstrate. In embodiments wherein a spacer strip is used and comprisesan adhesive material, the spacer may also provide means to hold thesubstrate and cover together. Other means of holding the substrate andcover together may be used and include clips and clamps. According tosome embodiments, hydrophobic spacer materials are preferred.

As can be seen in FIG. 1, the spacer strips are disposed along thelongitudinal edges of facing surface 28 but are not included on thelateral edges of the facing surface 28. Thus, a gap is provided at thelateral edges of the assembled device and can be employed as an entranceopening and/or an exit vent.

When assembled, a sample of fluid medium, for example, an aqueous PCRsample, can be introduced to the flow-through channel by entering a gapat a lateral edge of the assembly. The sample flows through and fillsthe flow-through channel and the sample chambers, which are in fluidcommunication with the flow-through channel. Then, a displacing fluid iscaused to enter the flow-through channel through a gap at a lateral edgeof the assembly, and the displacing fluid flows through and fills thechannel displacing sample from within the channel but without displacingsample from the sample chambers. The result is a plurality of discrete,isolated portions of the sample, held by the sample chambers.

According to the embodiment of FIG. 1 and other embodiments, samplechambers having dimensions of a few microns in diameter and a fewmicrons in depth can be provided, and result in sample chamber volumesof about 10 picoliters or less. In embodiments wherein sample chambersare provided having diameters of about 0.5 millimeter and depths ofabout 0.05 millimeter, sample volumes of about 10 nanoliters can beachieved.

The flow-through channel may have a depth of about 0.1 to about 500microns, preferably from about 10 to about 100 microns.

FIG. 2 is an exploded view of an analytical assembly according to anembodiment of the present invention, shown in partial cutaway. Theassembly comprises a substrate 50 and a cover 52. In the embodimentdepicted, the substrate and cover comprise substantially rigid platessuch as microscope slides, but may comprise more flexible materials suchas microscope slide coverslips. The substrate includes a bottom plate 53having an inner surface 54, and a patterned layer 56 of a first materialdisposed on the inner surface. The first material may be a patternedlayer comprising a hydrophobic material, and provides the substrate 50with a facing surface 58 which faces the cover 52. Within the patternedlayer 56 of the first material are formed a plurality of wells or holestherethrough defining bottom portions 60 of sample chambers. Each samplechamber bottom portion 60 has a closed lower end 62, defined by theinner surface 54 of the bottom plate 53. Each sample chamber also has asidewall which extends from the closed lower end 62 up to be facingsurface 58 of the substrate.

The cover 52 comprises a top plate 64 and a facing surface 66 whichcomprises a patterned layer 68 of a second material. The second materialmay be the same as the first material, and in some embodiments ispreferably moderately hydrophobic, that is, it preferably has a surfaceenergy of from about 30 dynes/cm to about 50 dynes/cm. The patternedlayer 68 may be smooth and of uniform thickness or, as shown in FIG. 2,the patterned layer 68 may have a plurality of upper portions 69 ofsample chambers formed therein which are complementary to the bottomportions 60 formed in patterned layer 56.

When assembled, the substrate 50 and cover 52 are attached together,with the facing surface 66 of the cover being spaced from the facingsurface 58 of the substrate. The space between the facing surfaces 66and 58 defines one or more flow-through channels through which samplefluid and displacing fluid may travel. Entrance openings 70 in the formof holes through the cover 52 are provided for the sample fluid anddisplacing fluid to enter the flow-through channel. Two entranceopenings 70 are provided as the assembly depicted in FIG. 2 comprisestwo flow-through channels. An exit vent (not shown) is provided for eachflow-through channel and may comprise a hole formed through thesubstrate or cover and in communication with the flow-through channel.The facing surfaces 66 and 58 are maintained spaced apart by spacerstrips 72, which may comprise transfer adhesive strips or patternedlayer applied to edge regions of the substrate. In embodiments wherein aspacer strip is used and comprises an adhesive material, the spacer mayalso provide means to hold the substrate and cover together. Other meansof holding the substrate and cover together may be used and includeclips and clamps. According to some embodiments, hydrophobic spacermaterials are preferred.

According to some embodiments of the invention, for example, someembodiments similar to that of FIG. 2, a centrally located entranceopening in the top plate can be provided and a sample fluid and/ordisplacing fluid can be loaded into the assembly by capillary force,centrifugal force, or other loading techniques.

As can be seen in FIG. 2, the spacer strips 72 are disposed along theentire peripheral edge of facing surface 58. A spacer strip 74 may alsobe included to divide the assembly into different portions. As shown inFIG. 2, spacer strip 74 divides the assembly into first and secondhalves, 76 and 78, respectively.

When assembled, the lower and upper portions of the sample chamberscomplement one another to form a plurality a sample chambers, eachhaving a closed lower end, a closed upper end, and a communication to aflow-through channel, the communication of each chamber being formed inthe sidewall of the chamber. A sample of fluid medium, for example, anaqueous PCR sample, can be introduced to the flow-through channelsthrough entrance openings 70. The sample flows through and fills theflow-through channels and both the lower and upper portions of thesample chambers, which are in fluid communication with the respectiveflow-through channels. Then, a displacing fluid is caused to enter theflow-through channels through the entrance openings 70, and thedisplacing fluid flows through and fills the channels displacing samplefrom within the channels but without displacing sample from the samplechambers. The result is a plurality of discrete, isolated portions ofthe sample, held by the sample chambers. According to some embodimentsof the invention, two different sample fluids are used, one in each half(76, 78) of the assembly.

FIG. 3 is a cross-sectional view through a longitudinal central portionof an analytical assembly according to another embodiment of the presentinvention. The assembly of FIG. 3 comprises a substrate 90 and a cover92. In the embodiment depicted, the substrate and cover comprisesubstantially rigid plates such as microscope slides, but may comprisemore flexible materials such as films or microscope slide coverslips.The substrate includes a bottom plate 93 having an inner surface 94, anda patterned layer 96 of a first material disposed on the inner surface.The first material may be a patterned layer comprising a hydrophobicmaterial, and provides the substrate 90 with a facing surface 98 whichfaces the cover 92. Within the patterned layer 96 of the first materialare formed a plurality of wells or holes therethrough defining bottomportions 100 of sample chambers. Each sample chamber bottom portion 100has a closed lower end 102, defined by the inner surface 94 of thebottom plate 93. Each sample chamber also has a sidewall which extendsfrom the closed lower end 102 up to the facing surface 98 of thesubstrate.

The cover 92 comprises a top plate 104 and a facing surface 106 whichcomprises a patterned layer 108 of a second material. The secondmaterial may be the same as the first material, and in some embodimentsis preferably moderately hydrophobic, that is, it preferably has asurface energy of from about 20 dynes/cm to about 30 dynes/cm. Thepatterned layer 108 may be smooth and of uniform thickness or, as shownin FIG. 3, the patterned layer 108 may have a plurality of upperportions 109 of sample chambers formed therein which are complementaryto the bottom portions 100 formed in patterned layer 96.

The substrate 90 and cover 92 are attached together, with the facingsurface 106 of the cover being spaced from the facing surface 98 of thesubstrate. The space between the facing surfaces 106 and 98 defines oneor more flow-through channels 110 through which sample fluid anddisplacing fluid may travel. An entrance opening 112 in communicationwith the flow-through channel 110, in the form of a gap formed at alateral end of the assembly, is provided for the sample fluid anddisplacing fluid to enter the flow-through channel 110. An exit vent 114is provided for sample to exit the channel 110 as the sample isdisplaced from the channel by the displacing fluid. The entrance openingand exit vent may instead be in the form of holes formed through thesubstrate and/or cover. The facing surfaces 106 and 98 are maintainedspaced apart by spacer strips 116, which may comprise transfer adhesivestrips or a patterned layer applied to edge regions of the substrate. Inembodiments wherein a spacer strip is used and comprises an adhesivematerial, the spacer may also provide means to hold the substrate andcover together. Other means of holding the substrate and cover togethermay be used and include clips and clamps.

As can be seen in FIG. 3, the spacer strips are disposed along thelongitudinal edges between the facing surfaces but are not included onthe lateral edges of the of facing surfaces. Thus, gaps used as theentrance opening and exit vent are provided at the lateral edges of thedevice.

As can be seen in FIG. 3, the lower portions 100 and upper portions 109of the sample chambers complement one another to form a plurality asample chambers 118, each having a closed lower end, a closed upper end,and a communication to a flow-through channel, the communication of eachchamber being formed in the sidewall of the chamber. A sample of fluidmedium, for example, an aqueous PCR sample, can be introduced to theflow-through channel 110 through entrance openings 112. The sample flowsthrough and fills the flow-through channel and both the lower and upperportions of the sample chambers. Then, a displacing fluid is caused toenter the flow-through channel through the entrance openings, and thedisplacing fluid flows through and fills the channel displacing samplefrom within the channel but not displacing sample from the samplechambers. The result is a plurality of discrete, isolated portions ofthe sample, held or retained by the sample chambers. The sample fluidretained in the sample chambers may form a cylinder extending from theclosed lower, through and interrupting the flow-through channel, and upto the closed upper end.

FIG. 4 is a perspective view of a bottom portion of an analyticalassembly according to an embodiment of the present invention, with thecover removed. The bottom portion of the assembly comprises a substrate120, depicted in the figure as a coated substantially rigid plate suchas microscope slide. The substrate includes a bottom plate 122 having aninner surface 124, and a patterned layer 126 of a first materialdisposed on the inner surface. The patterned layer has a substantiallyuniform thickness. The first material may comprise a hydrophobicmaterial, and provides the substrate 120 with a facing surface 128 whichfaces a cover (not shown), for example, the cover used in the embodimentof FIG. 1. On the patterned layer 126 are formed a plurality of patches130 of sample retaining material, preferably capable of retaining afluid sample volume of from about 1 microliter to about 1 picoliter orless. Adhesive strips, a patterned layer of pressure sensitive adhesive,or a patterned layer of a curable adhesive, 132, may be used to attach acover to the bottom portion.

According to some embodiments, the patch occupies a volume of about 100nanoliters or less. The patch may be an absorbent material which absorbsand retains sample, or the patch may be made of material which has anaffinity sufficient enough to retain, for example, an aqueous PCRsample. According to some embodiments, the patches of material comprisea hydrophilic material. According to embodiments of the inventionwherein the sample retaining patches comprise a substantiallynon-absorbent material which has a retaining affinity for a sample, thepatch defines a closed lower end of a sample chamber defined between theexposed surface of the patch and the facing surface of a cover. In someembodiments, a patch of porous material may fill a volume between thesubstrate and the cover and the sample chamber is defined by the voidvolume of the porous patch.

FIG. 5 is a cross-sectional view through a longitudinal central portionof an analytical assembly according an embodiment of the presentinvention wherein sample chambers comprise absorbent material patches orcomprise the volume between complementary opposing patches of sampleretaining materials.

The assembly of FIG. 5 comprises a substrate 134 and a cover 136attached to one another. In the embodiment depicted, the substrate andcover comprise substantially rigid plates such as microscope slides. Thesubstrate includes a bottom plate 138 having an inner surface 140, and apatterned layer 142 of a first material disposed on the inner surface.The first material may be a hydrophobic material, and provides thesubstrate 134 with a facing surface 144 which faces the cover 136. Onthe patterned layer 142 are formed a plurality of sample retainingpatches 146 which define sample chambers by absorbency or surface energyor other retentive property.

The cover 136 comprises a top plate 148 and a patterned layer 150 of asecond material defining a facing surface 152. The second material maybe the same as the first material, and in some embodiments is preferablya hydrophilic material if aqueous samples are to be retained andisolated. The patterned layer 150 may be smooth and of uniform thicknessor, as shown in FIG. 5, the patterned layer 150 may have a plurality ofsample retaining patches 154 complementary to and mirroring patches 146.

The substrate 134 and cover 136 are attached together, with the facingsurface 152 of the cover being spaced from the facing surface 144 of thesubstrate. The space between the facing surfaces 152 and 144 defines oneor more flow-through channels 156 through which sample fluid anddisplacing fluid may travel. An entrance opening 158 comprising a holeformed through the cover and in communication with the flow-throughchannel 156. An exit vent 160 comprising a hole through the substrate134 is provided for sample to exit the channel 156 as the sample isdisplaced from the channel by the displacing fluid.

The facing surfaces 152 and 144 are maintained spaced apart by spacerstrips 162, which may comprise transfer adhesive strips applied to edgeregions of the substrate. In embodiments wherein a spacer strip is usedand comprises an adhesive material, the spacer may also provide means tohold the substrate and cover together. Other means of holding thesubstrate and cover together may be used and include clips and clamps.

According to some embodiments, the patches comprise an absorbent sampleretaining material. According to some embodiments, the patches retainsample by surface energy, and have an affinity for a sample, forexample, a hydrophilic material patch which retains an aqueous sample ona surface 164 thereof. In some embodiments, the affinity may be inducedby optical or electromagnetic means. The sample chamber may comprise thevolume 166 between the facing surfaces of each complementary pair ofpatches, wherein the sample chambers have no sidewalls but are definedas the volume between the two complementary patch surfaces. The affinityof the two opposing patch surfaces to a retained sample is sufficient tosupport a column of sample between the two patches while a displacingflows through the channel adjacent the column. In some embodiments,patches of porous material extending from the substrate to the cover mayinterrupt the flow-through channel and the sample chamber may be definedas the void volume of a porous patch.

Sample flows through and fills the flow-through channel and the samplechambers, which are in fluid communication with the flow-throughchannel. A displacing fluid can subsequently be caused to enter theflow-through channel through the entrance opening, displacing samplefrom within the channel but not displacing sample from the samplechambers. The result is a plurality of discrete, isolated portions ofthe sample, held or retained or absorbed by the sample chambers.

FIG. 6A is a perspective view of another embodiment of the presentinvention, and FIG. 6B shows an enlargement of portion VIB from FIG. 6A.As can be seen in FIGS. 6A and 6B, a bottom support plate 174 supports asample retaining means 167 comprising a plurality of pores or cavities168 having open ends in communication with an exposed surface 170 onwhich a sample of fluid is applied. The pores have a first affinity to asample of fluid medium such that when the sample is disposed upon theexposed porous surface 170 the sample is drawn into and retained by theplurality of pores 168. For example, through capillary or other force,the sample may wet and fill the pores 168. The pores 168 havesubstantially the same volume as one another and each has a closed lowerend 169. Preferably the pores 168 each have a volume of about 1 μl orless, more preferably, about 10 nl or less, and for some applicationsabout 1 nl or less. The closed lower ends 169 of the pores are definedor bound by a layer of coating material 172 deposited on a bottom plate174. The coating material may be a hydrophilic material if aqueoussamples are to be retained in the pores.

A cover 171 is provided on one of the sample retaining means shown inFIG. 6A. The cover 171 can be used to displace sample fluid from theporous surface 170 of the retaining means and for sealing sample withinthe pores 168. The cover 171 displaces sample fluid from the poroussurface without displacing sample from, within the pores. Preferably,the cover 171 comprises a hydrophobic coating 173 on the surface of thecover which contacts the exposed surface 170 of the sample retainingmeans. The coating 173 contacts sample and can be used to squeeze excesssample off of the surface 170. A means for sealing the sample of fluidwithin the pores is also provided, and in the embodiment shown in FIG.6B the means for displacing excess sample and the means for sealingsample in the pores are the same means, that is, the cover 171.Preferably, the coating 173 is adhesive as well as hydrophobic, althoughan adhesive and/or hydrophobic coating may instead or additionally beprovided in the exposed surface 170 of the sample retaining means.Sealing the sample in the pores prevents evaporation and contaminationof the sealed sample.

The displacing means and the sealing means, for example the cover 170shown in FIGS. 6A and 6B, may be pressed against the exposed surface 170by any of a variety of means. Clamps, clips or springs may be used toattach the sealing means to the exposed surface 170, and/or to force thedisplacing means against the exposed surface.

According to some embodiments of the invention, other means may be usedas displacing means for a device similar to, or the same as, that shownin FIGS. 6A and 6B. For example, according to some embodiments, a dropor bead of curable fluid, for example, a curable adhesive, can be forcedacross an exposed surface of a sample retaining means having one or moresample retaining cavity, recess, hole or pore formed in the surface.Gravity, pressurized gas, or mechanical means, for example, can be usedto force the curable fluid across the exposed surface, displacing excesssample from the surface without displacing sample from within the sampleretaining cavity. According to some embodiments of the invention, as thecurable fluid traverses the exposed surface a thin layer of the fluid isdeposited on the surface and coats the top of a sample portion retainedin the cavity. Upon curing, the curable fluid forms a seal for thesample retained within the cavity such that the sample retained isisolated from other cavities and from excess sample. The sealed sampleis protected from contamination and evaporation. Preferably, the curablefluid seal is sufficient to prevent contamination and evaporation of theentrapped sample during thermal cycling conditions generally used in PCRamplification methods.

According to some embodiments of the invention, separate displacingmeans and sealing means are provided for isolating a sample portionwithin a sample retaining cavity. For example, according to embodimentsof the invention, a displacing means is provided for removing excesssample from an exposed surface of a device having a sample retainingcavity associated therewith. The sample retaining cavity may be formedon, formed in, in contact with or adjacent to a substrate. The cavitymay be defined by at least one sidewall, and the sidewall may comprise ahydrophilic material. The displacing means may comprise a wiping devicesuch as a squeegy, a wiper, a blade or other scraping or rubbing devicewhich can physically move excess sample away from the open upper end ofa sample retaining cavity and preferably off of the exposed surface ofthe sample retaining means. Preferred displacing means may comprise awiping device made of an elastomeric Material, for example, a stiffsilicone rubber blade. Preferably, the wiping device comprises ahydrophobic material to which aqueous sample fluids will not cling.

According to some embodiments, the displacing means comprises an openingor channel through which a sample retaining means snugly fits, and awiping device is provided adjacent to the opening or channel such thatthe wiping device wipes excess sample from an exposed surface of thesample retaining means as the retaining means is forced through theopening or channel.

According to some embodiments of the invention, sealing means areapplied to the sample retaining cavity immediately after the displacingmeans displaces excess sample from adjacent the sample retaining cavity.When retained sample portions having volumes of about 1 μl or less areformed, they tend to evaporate rapidly and thus require prompt sealingto protect sample integrity. According to some embodiments of theinvention, a wiping device is used to displace excess sample fluid froman exposed surface of a sample retaining means, and the wiping device isprovided with a trailing edge that pulls a bead or drop of a curablesealing fluid across the exposed surface as a leading edge of the wipingdevice displaces excess sample.

Example 1 and Control 1

Conventional PCR was performed in 0.2 ml polypropylene ependorf tubes toset standards. Microcapillary PCR was then performed according to thepresent invention on the same sample material in quartz glassmicrocapillaries. The PCR sample containing the nucleic acid sequence tobe amplified was prepared and included materials from a “TaqMan” kitavailable from Perkin-Elmer, Applied Biosystems Division, Foster City,Calif. The kit contained human DNA at 10 ng/μl, the forward primer5′-TCACCCACACTGTGCCCATCTACGA-3′ (SEQUENCE ID NO:1) and the reverseprimer 5′-CAGCGGAACCGCTCATTGCCAATGG-3′ (SEQUENCE ID NO:2) that amplify a295 bp segment of the human B actin gene, and a dual fluor-labeled probecomprising 5′-[6FAM]-ATGCCC-[TAMRA]-CCCCCATGCCATCCTGCGT-3′ (SEQUENCE IDNO:3) that is complementary to bases 31 to 56 of the PCR product. Thedesignation FAM represents 6-carboxyfluorescein, and TAMRA represents6-carboxytetramethyl-rhodamine. With reference to the Control 1 andExample 1, the term “FAM” is referred to herein as “fluorescein” and theterm “TAMRA” is referred to herein as “rhodamine”.

The PCR sample comprised Taq polymerase available from BoehringerMannheim, Indianapolis, Ind., and anti-Taq antibody from Clonetech, PaloAlto, Calif. The sample also comprised concentrations of 10 mM Tris-HCl(pH 8.3), 50 mM KCl, about 0.01% by weight gelatin, 500 μg/ml to 5 mg/mlbovine serum albumin (BSA), 3.5 mM MgCl₂, 0.2 mM each of dATP, dCTP,dGTP and dUTP, 0.3 μM forward and reverse primers, 0.2 μMdual-fluor-labeled probe, 0.5 manufacturer's units (u) Taq polymeraseper 10 μl PCR mixture, 0.1 μl anti-Taq antibody per 10 μl PCR mixture,and varying amounts of template DNA. The specific activity of Taqpolymerase was about 250,000 units per mg which, at a molecular weightof 100,000 Daltons, translates to about 10⁹ molecules per μl. Since Bactin is a single copy gene, it was estimated that there is one copy ofβ actin template per 3 pg of human genomic DNA. In some PCRs thedual-fluor-labeled probe was replaced with the fluorescent, DNA-stainingdye SYBR™ Green I (product #S-7567) available from Molecular. Probes,Eugene, Oreg., used a 10⁻⁴ dilution from the stock supplied by themanufacturer.

The conventional PCRs (CONTROL 1) performed in the 0.2 ml polypropylenetubes were thermocycled in a model 9600 thermocycler from Perkin Elmerusing 92° C. for 15 seconds, 54° C. for 15 seconds, and 72° C. for 15seconds, for 40 cycles.

Microcapillary PCR was performed according to the present invention inquartz glass microcapillaries from Polymicro Technologies (Phoenix,Ariz.). These capillaries had inner diameters ranging from 20 μm to ˜75μm and outer diameters of 250 μm to 375 μm. The capillaries come witheither a polyimide or Teflon external coating to make them flexible.Because the polyimide coating is opaque and fluorescent, it had to beremoved before use. The coating was removed by flaming a segment ofpolyimide capillary with a Bunson burner for several seconds and thengently wiping off the burned coating. Flaming and wiping was repeated asnecessary until the capillary was clear. The resulting bare capillarieswere very fragile. The Teflon coated microcapillaries were easier towork with since the optically clear and non-fluorescent Teflon coatingdid not need to be removed. Both types of capillaries gave equivalentresults in PCR.

Hundreds of microcapillary tubes (EXAMPLE 1) were filled by touching anopen end to a drop of the PCR sample which wicked in by capillaryaction. The microcapillaries were then sealed and supported by gluingthe two ends thereof to two respective coverslips, leaving anunsupported segment in the middle, thus minimizing thermal mass. AUV-curable fluid glue was used to seal the end of the tube and to gluethe tube ends to coverslips. Assemblies comprising a plurality ofmicrocapillaries were formed by sealing the ends of each tube in theassembly to the same pair of coverslips.

The glue was available as optical adhesive #81 from Norland Products,New Brunswick, N.J. The glue was cured by exposure to 366 nm UV light. Asource of 366 nm wavelength light is UV lamp model UVL-21, availablefrom UVP Inc., San Gabriel, Calif. The lamp was held about 1 cm from thesample for 30 seconds. The PCR mixture was shielded from UV light bylaying a small piece of opaque paper over the center section of thecapillary during UV exposure.

FIG. 7 is a top plan view of an exemplary microcapillary device used inaccordance with Example 1 of the present invention. As shown in FIG. 7,an assembly 176 is provided having four microcapillary tubes 178 havinginner diameters of about 100 microns each, and two support tubes 180having outer diameters of about 1.5 mm. The ends of the tubes 178 and180 were adhered to two respective plates or bases 182 with UV-curableadhesive 184. One end of each tube microcapillary and support tubeshared a respective plate 182. In the embodiment shown in FIG. 7, theplates comprised standard 1″×1″ microscope cover slips and the tubeswere each about 4 cm long. All the tubes were arranged substantiallyparallel to one another with the microcapillary tubes 178 each beingpositioned between the two larger diameter support tubes.

The sample holding assembly 176 was attached with SCOTCH tape to thesample holder of a Rapidcycler air oven available from IdahoTechnologies, Idaho Falls, Id., and cycled through a protocol comprisingcycles of 92° C. for 5 seconds, 54° C. for 5 seconds, and 72° C. for 15seconds, for 40 cycles. The cycling protocol took about 30 minutes inthe Rapidcycler.

After PCR cycling, fluorescence of the samples was measured with a ZeissAxiovert 410 laser scanning microscope using a 20X-0.5NA objective, 15mW external argon laser approximately 5% of the power of whichwas'focused to a spot size of 1 μm², and band pass filters of 515-565 nmfor fluorescein and SYBR™ Green, and >590 nm for rhodamine (power lossand spot size estimates provided from the manufacturer). Average pixelintensity was measured in regions of about 20 μm×50 μm overlying thecapillary age. Fluorescence intensity was examined visually along the2.5 cm lengths of capillaries by manually translating the stage;quantitative measurements were made every 2-5 mm or more often ifvariability in the fluorescence signal was observed.

Results

When PCRs were performed in 20 μl volumes in ependorf tubes in a model9600 Thermocycler (Perkin Elmer, Norwalk, Conn.), the B actin primersamplified an approximately 300 bp segment from human DNA as expected.PCRs performed in the presence of the TaqMan probe were transferred tocapillary tubes and analyzed by fluorescence microscopy. Typical valuesfor average pixel intensity were about 130 (relative fluorescence units)for fluorescein and about 60 for rhodamine, with values of backgroundemission from empty capillaries of about 20 at both wavelengths. Indifferent experiments the fluorescein:rhodamine (F/R) intensity ratiovaried from about 1.0:1.0 to about 2.0:1.0 in samples containing PCRproduct. For negative control PCRs containing no template DNA, no Tagpolymerase, or no reverse primer, the rhodamine emission was about thesame (about 60), while the fluorescein emission was reduced to about 30,giving a F/R intensity ratio of about 0.5. The absolute values offluorescein and rhodamine emission varied between experiments and withsmall changes in machine settings (laser power, attenuation, brightness,contrast) whereas the F/R intensity ratio was fairly constant.Therefore, the F/R intensity ratio was used as a measure of whether theβ actin product had been amplified.

The yield of PCR product in conventional reactions in polypropylenetubes was estimated by ethidium bromide staining of product in agarosegels, and by adding a known amount of ³²P-dCTP to a PCR and countingradioactivity in the purified PCR product. Both methods gave an estimateof about 10¹¹ product molecules/μl of PCR. This corresponds to a productconcentration of about 0.16 μM, which implies that about half of the PCRprimers were converted to product.

To assess the extent of degradation of TaqMan probe following PCR, theeffect of mung bean nuclease on the F/R intensity ratio was examined.Treatment of probe with mung bean nuclease for 10 minutes at 37° C.raised the F/R intensity ratio from 0.5 to 5. This presumably representscomplete degradation since further incubation did not increase theratio. An F/R intensity ratio of 1.5, characteristic of positive PCRs,therefore suggests that about 30% of probe was degraded. Thiscorresponds to a concentration of degraded probe of 0.06 μM and impliesthat about one third of the probe that could have hybridized to PCRproduct was degraded.

When PCRs were performed in small diameter glass capillaries, the volumeof the reaction was too small to detect PCR product by standard gelelectrophoresis. While products might have been detectable by capillaryelectrophoresis, it was of interest to see whether the TaqMan assaycould be used as a detection method. The F/R intensity ratio wastherefore used as a surrogate measure of amplification. This ratio wasabout 0.5 in negative control reactions (no template, no enzyme, or noreverse primer) and was usually greater than 1 in samples where productwas expected. In capillaries containing terminal dilutions of genomicDNA template, the ratio sometimes varied with position along thecapillary, which was attributed to localized accumulation of degradedprobe, discussed in more detail below. In cases where the intensityratio varied, the maximum value of the F/R intensity ratio in thecapillary was used as the measure of whether the target sequence hadbeen amplified.

A histogram of the maximum values of the F/R intensity ratio in over 100capillary reactions of terminally diluted genomic DNA is shown in FIG.8, along with the corresponding values for negative control reactions.The negative controls had a mean ratio of 0.5 with a range of 0.4 to0.9. The experimental samples had a bimodal distribution with one aim ofthe distribution paralleling that of the negative control samples. Thissuggests that the experimental samples consisted of positive andnegative samples. Since the nadir of the experimental sampledistribution occurred at about an F/R intensity ratio of 1, we chosethis value as a “cut-off” to distinguish positive from negative samples.This “cut-off” is consistent with the F/R values of 1 to 2 in PCRscarried out in conventional volumes in ependorf tubes.

Using a “cut-off” of F/R≧1, the sensitivity of the detection system wasestimated by mixing exonuclease-digested probe with undegraded probe. AnF/R ratio of ≧1 was obtained when ≧0.02 μM degraded probe was mixed with0.2 μM undegraded probe. This corresponds to about 10⁸ molecules ofdegraded probe in a 10 nl volume. Using the confocal feature of themicroscope, it was determined that the signal decreased rapidly when thedepth of field dropped below 20 μm. Thus, an estimate of the lower limitof detection for this system is about 10⁵ molecules of degraded probe ina volume of 20 μm×20 μm×20 μm, that is, about 10 picoliters (pl).

As has been noted by others performing PCR in glass tubes, for examplein the publication of Wittwer et al., Rapid Cycle DNA Amplification:Time and Temperature Optimization, BioTechniques, Vol. 10, pp. 76-83(1991), it was important to include bovine serum albumin (BSA) in thePCRs. Presumably, BSA blocks non-specific sticking of DNA to glass. WhenBSA was not included, the F/R intensity ratio was about 0.5. Generally,a 500 μg/ml final concentration of BSA was used in the PCRs although forsome batches of BSA the concentration was increased to 5 mg/ml.

Human DNA was diluted so that PCRs contained 0-14 haploid genomeequivalents (0-42 pg)/capillary. Reactions were scored as positive ifthe maximum F/R intensity ratio along the tube was 1.0 or greater. Theresults for a series of PCRs in capillaries with internal diameters of20 to 75μ are shown in Table 1 below and shown graphically in FIG. 8.

TABLE 1 Replicate PCRs in microcapillaries with terminal dilutions ofgenomic DNA Haploid Probability of Capillary genome ≧1 template Fractiondiameter equivalents per capillary positive Number of (microns) percapillary, m 1-e^(−m) PCRs PCRs 20 0 0.00 0.00 2 0.2 0.18 0.10 10 0.50.39 0.33 15 25 0 0.00 0.00 4 0.4 0.33 0.28 18 0.8 0.55 0.50 8 1.5 0.781.00 3 3 0.95 1.00 3 30 0 0.00 0.00 8 0.5 0.39 0.52 23 1 0.63 0.92 131.5 0.78 1.00 10 4 0.98 1.00 3 50 0 0.00 0.00 13 0.4 0.33 0.00 3 0.80.55 0.67 27 1.5 0.78 0.82 28 3 0.95 1.00 2 6 1.00 0.89 9 13 1.00 1.00 775 0 0.00 0.00 7 0.8 0.55 0.50 6 1.7 0.82 0.67 12 3.4 0.97 1.00 9 7 1.001.00 5 14 1.00 1.00 6

Capillaries containing more than one haploid genome equivalent generallyhad F/R intensity ratios greater than 1. In capillaries containing lessthan one haploid genome equivalent, the fraction of capillaries with F/Rintensity ratio ≧1 was roughly proportional to the fraction ofcapillaries expected to contain 1 or more template molecules. Thisfraction was calculated from the Poisson distribution as 1−e^(−m) wherem=the amount of DNA/capillary/3 pg. The results provide strong supportfor the hypothesis that reactions were positive when capillariescontained 1 or more template molecules.

Example 2

Similar results were obtained with another preparation of human genomicDNA obtained from Promega: at 8 haploid genome equivalents (24 pg) percapillary, 4 of 4 capillaries gave maximum F/R intensity ratios ≧1; at0.7 haploid genome equivalents (2 pg) per capillary, 3 of 4 capillarieswere positive; at 0.1 haploid genome equivalents (0.4 pg) per capillary,0 of 4 capillaries were positive.

Example 3

The inhomogeneity of F/R intensity ratio along the length of capillariescontaining about 1 template molecule suggested that residuallocalization of degraded probe may be observed as a result of localizedaccumulation of PCR product. To investigate this possibility,amplifications in 2.5 cm long sections of capillaries containing about0.5 haploid genome equivalent per capillary were performed. A plot ofF/R intensity ratio along a few representative capillaries is shown inFIGS. 9-14. Some capillaries had a single peak while others had two. Twopeaks indicate two areas where PCR product and degraded probe hadaccumulated. The half-widths of the peaks (measured at half-height) wereabout 3-6 mm. When capillaries were left overnight, the distributionsbroadened and flattened. Inhomogeneities in F/R intensity ratio were notseen when capillaries were examined before PCR, or after PCR incapillaries containing no template DNA or about 75 initial templatemolecules. Representative experiments are shown in Table 2 below. Theresults indicate that high variability in the F/R intensity ratio isspecific for capillaries with about 1 target molecule and decreases withtime.

TABLE 2 NUMBER OF HAPLOID CAPILLARIES STANDARD GENE WITH AVERAGEDEVIATION EQUIVALENTS TIME WHEN MAXIMUM F/R F/R OF F/R PER FLUORESCENCENUMBER OF INTENSITY INTENSITY INTENSITY GROUP CAPILLARY ANALYZEDCAPILLARIES RATIO >1 RATIO RATIO A 0  1 HOUR AFTER PCR 9 0 0.50 0.05 B75  1 HOUR AFTER PCR 10 10 1.75 0.10 C 1 BEFORE PCR 5 0 0.51 0.04 D 1  1HOUR AFTER PCR 5 4 0.83 0.46 E 1 24 HOURS AFTER PCR 5 3 0.89 0.23 F 1 48HOURS AFTER PCR 5 1 0.82 0.23 G 1 BEFORE PCR 5 0 0.46 0.03 H 1  1 HOURAFTER PCR 5 5 1.22 0.43 I 1 24 HOURS AFTER PCR 5 5 1.14 0.25 J 1 48HOURS AFTER PCR 5 5 1.11 0.21

These results support a theory that the inhomogeneities were not due tosmudges blocking light transmission, thermal variations during PCR, orphotobleaching. It was also determined that the 30 seconds of UVirradiation used to cure the sealing glue at the ends of the capillariesdid not alter the F/R ratio. Photobleaching of fluorescein (but not therhodamine) was detectable with repeating laser scanning at the highestpower, with 10 scans reducing the fluorescein signal about 10%; however,only 1 or 2 scans at this power were performed at any one location whencollecting data, and thus photobleaching does not explain theinhomogeneities. To see if convection after PCR might be broadeningpeaks, 0.2% by weight to about 0.8% by weight low-melt agarose was addedto some PCRs but no effect of the agarose was noted. A few of thecapillaries fortuitously contained air bubbles that divided the sampleinto two or more segments. In several of these cases, the F/R intensityratio was ≧1 on one side of a bubble and 0.5 on the other side,consistent with blocked diffusion of degraded probe.

To confirm the results of the TaqMan assay, the fluorescent dye SYBR™Green I was substituted for the TaqMan probe. Because the fluorescenceof SYBR™ Green I increases many fold in the presence of double strandedDNA, it can be used to detect double stranded PCR product, although itdoes not distinguish spurious product such as “primer dimer” fromdesired product. The SYBR™ Green I fluorescence assay has to beperformed at elevated temperature to reduce background fluorescence fromnon-specific annealing of primers. To do this, segments of capillarieswere placed, after PCR, in about 1 ml of mineral oil in a special 35 mmpetri dish, the bottom of which was made of optically clear, conductingglass coated with a thin layer of indium tin oxide available fromBioptechs, of Butler, Pa. By applying 3-4 volts across the bottom of thedish, the temperature in the oil was raised to about 70° C. Because onlya portion of the bottom of the petri dish was flat and accessible in themicroscope, the capillaries had to be cut after PCR into approximately 1cm segments in order to be imaged.

Using this device, PCR product derived from single template moleculescould be detected. For example, the fluorescence intensity was 155-194in 7 capillary segments derived from a PCR containing 30 haploid genomeequivalents per cm of capillary length, compared to a fluorescenceintensity of 40-57 in 7 capillary segments containing no template DNA.The variability in fluorescence at 2-3 mm intervals along thesecapillaries was about 20%. In contrast, in 7 capillary segments derivedfrom PCRs containing 0.3 haploid genome equivalents per cm of capillary,the fluorescence intensity varied from 49 to 136, with 4 capillarysegments having fluorescence intensity <73 at all tested positions alongtheir lengths, 2 capillary segments having fluorescence intensity >100at all positions, and one capillary having a fluorescence intensity of68 at one end increasing to 122 at the other end thereof. These resultsprovide additional evidence that PCR products derived from singlemolecules can be detected and remain localized in microcapillaries forseveral hours after PCR.

Example 4

A device substantially similar to that depicted in FIG. 3, having asubstrate and a cover comprising microscope slide coverslips, andprovided with first and second patterned layers comprising cured Norland68 UV-curable adhesive coated respectively thereon by screen printing,was filled by capillary action with a Beta-actin polymerase chainreaction solution. The substrate and coverslip were spaced apart andheld together with adhesive strips and the first and second patternedlayers had complementary sample chamber portions formed therein havingradii of about 1 mm. A displacing fluid comprising uncured Norland 81UV-curable adhesive was then loaded into the flow-through channel bycapillary action and displaced the polymerase chain reaction solutionfrom the flow-through channel but not from the sample chambers. Thedevice was then exposed to UV light to cure the displacing fluid, andthe sample portions were shielded from the light with a transparencylaid on top of the device and having inked spots aligned with the samplechambers. The device was then thermocycled and analyzed by fluorescencemicroscopy as described in connection with Example 1. Six of six samplechambers estimated to contain an average of 3 copies of genomic DNAtemplate had F/R intensity ratios greater than 1 and four of six samplechambers estimated to contain an average of 0.3 template copies had F/Rintensity ratios of greater than 1.

Discussion

The results presented above provide strong evidence that the TaqManassay can easily detect as little as 1 template molecule when the volumeof the reaction is on the order of 10 nl. For 50 μm inner diametercapillaries, the reaction volume is about 20 nl per cm length ofcapillary. Using terminal dilutions of two preparations of genomic DNA,good correlation has been found according to the invention between thenumber of capillaries giving positive reactions and the number ofcapillaries calculated to contain 1 or more template molecules. Theinference of single molecule sensitivity is further supported by theobservation of peaks of elevated F/R emission along the length ofcapillaries estimated to contain 1 or 2 template molecules. Presumablythese peaks results from localized accumulation of PCR product andcorresponding degraded probe.

The localized accumulations of PCR product and degraded probe remaineddetectable for several hours after PCR. It is believed that thenarrowness of the capillaries effectively eliminates convection so thatmolecular movement is dominated by diffusion. Molecules the size ofcompletely degraded probe (e.g. rhodamine-dGTP) have diffusion constantsof about 3-5×10⁻⁶ cm²/sec in water at room temperature as reported inthe publication of Chang, PHYSICAL CHEMISTRY with application toBiological Systems, MacMillan Publishing Co., New York, page 87. Thediffusion constant increases with temperature as D is proportional tokT/η, where T is measured in degrees Kelvin and η, the viscosity,decreases with temperature. The viscosity of water decreases about3-fold as temperature increases from 25° C. (298° K.) to 92° C. (365°K.); thus, D would increase about 3.25-fold over this temperature range.The root mean square distance traveled by a molecule with diffusionconstant D in time t is (2Dt)^(1/2) or, about 2-5 mm in 2 hours formolecules the size of completely degraded probe at temperatures between25° C. and 92° C. The PCR product, based on its molecular weight, shouldhave a diffusion constant of about 0.45×10⁻⁶ cm²/sec and should diffuseabout 3 times less far than degraded probe in the same time. Thesecalculations indicate that the widths of the observed fluorescent peaksare consistent with diffusion-mediated spreading of PCR product anddegraded probe.

The width of peaks might be slightly larger than predicted by diffusion,due to the tendency of PCR to saturate in regions where theconcentration of PCR product is high. So long as all of the amplifiedmolecules are replicated each cycle, the progeny from a single startingtemplate will have the same average displacement (i.e., root mean squaredisplacement) as a collection of independent molecules, that is, theywill appear to diffuse with a root mean square displacement that isproportional to the square root of the time. However, as PCR begins tosaturate, molecules near the center of the distribution whereconcentration is high have a lower probability of being replicated thanmolecules near the “edge” of the distribution where concentration islow. This unequal probability of replication tends to make thedistribution broader.

The diffusion model suggests that detection of single target moleculesby TaqMan assay would be difficult using conventional size capillaries.A 5 mm segment (characteristic diffusion distance for degraded probe) ina 0.8 mm inner diameter capillary contains about 2.5 μl. After spreadingin 1/50th of this volume (about 50 nl), the fluorescent signal obtainedfrom single starting molecules in 50 μm inner diameter capillaries wassometimes not above the background (see Table 2, average F/R intensityat 24 and 48 hours and number of capillaries in which maximum F/R>1).Thus, PCR would have to be significantly more efficient than achieved todetect single molecules in volumes greater than 1 μl.

While limited diffusion of product and degraded probe was important forour ability to detect single starting molecules in capillaries,diffusion of reactants present in the original reaction mixture isusually not limiting for PCR; for example, at the conventionalconcentration of Tag polymerase used here, the average distance betweenpolymerase molecules is about 1 μm, and polymerase molecules (MW about100,000 Daltons) diffuse this distance in about 0.01 second. Thus, allportions of the reaction should be sampled by a polymerase molecule manytimes each second.

The data also shows that single DNA molecules or segments can bedetected with the “TaqMan” system when PCRs are confined to volumes of100 nanoliters or less, preferably 60 nanoliters or less, by usingcapillaries with small diameters and relying on the fortuitously slowrate of diffusion. Many PCR reactions with single molecule sensitivitycan be performed simultaneously in small spaces by confining PCR's tosmall regions in 3 dimensions as described in other embodiments of thepresent invention. The devices of the invention can be used to measurethe number of template molecules in a sample simply by counting thenumber of positive reactions in replicate PCRs containing terminaldilutions of sample. Due to the closed system environment which preventscarryover contamination, and the ability to automate fluorescencedetection, devices according to the present invention and methods forusing the devices have significant potential for clinical uses of PCR.An assay based on presence versus absence of PCR product in replicatereactions may be more robust with respect to small changes inamplification efficiency than quantitative competitive assays ortime-to-reach-threshold level assays that require assumptions aboutrelative or absolute amplification rates.

Although the present invention has been described in connection withpreferred embodiments, it will be appreciated by those skilled in theart that additions, modifications, substitutions and deletions notspecifically described may be made without departing from the spirit andscope of the invention defined in the appended claims.

What is claimed is:
 1. A device for amplifying target nucleic acid in asample, the device comprising: a planar fluidic assembly comprising: asubstrate, a porous material layer on a surface of the substrate, acover over the porous material layer and sealingly affixed to thesubstrate, wherein the cover is spaced from the porous material layer; aflow channel defined between the porous material layer and the cover,wherein the flow channel has a substantially uniform cross-section froma first end to a second end, an inlet in flow communication with thefirst end of the flow channel to introduce sample containing targetnucleic acid into the flow channel, an outlet in flow communication withthe second end of the flow channel, a plurality of nucleic acid primersretained by the porous material layer at discrete regions along andwithin the flow channel, each of the plurality of nucleic acid primersbeing complementary to a portion of the target nucleic acid in thesample to enable a primer-based amplification reaction of the targetnucleic acid, wherein the porous material layer is configured to retain,at the discrete regions and during the primer-based amplificationreaction, the sample introduced to the flow channel and amplifiedproduct of the amplification reaction.
 2. The device of claim 1, furthercomprising fluorescence-based detection reagents retained by the porousmaterial layer at the discrete regions.
 3. The device of claim 1,wherein the flow channel comprises a plurality of flow channels.
 4. Thedevice of claim 1, wherein the porous material layer comprises aplurality of individual porous patches located at the discrete regions.5. The device of claim 1, wherein the discrete regions are arranged in atwo-dimensional array.
 6. The device of claim 1, wherein the cover isspaced at a substantially uniform distance from the porous materiallayer.